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WO1999058658A2 - Vecteurs d'expression destines a stimuler une reponse immunitaire et procedes de leur utilisation - Google Patents

Vecteurs d'expression destines a stimuler une reponse immunitaire et procedes de leur utilisation Download PDF

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Publication number
WO1999058658A2
WO1999058658A2 PCT/US1999/010646 US9910646W WO9958658A2 WO 1999058658 A2 WO1999058658 A2 WO 1999058658A2 US 9910646 W US9910646 W US 9910646W WO 9958658 A2 WO9958658 A2 WO 9958658A2
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Prior art keywords
epitopes
peptide
epitope
ctl
htl
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PCT/US1999/010646
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English (en)
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WO1999058658A3 (fr
Inventor
John D. Fikes
Gary G. Hermanson
Alessandro Sette
Glenn Y. Ishioka
Brian Livingston
Robert W. Chesnut
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Epimmune, Inc.
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Priority to AU40785/99A priority Critical patent/AU4078599A/en
Priority to EP99924233A priority patent/EP1078092B1/fr
Priority to JP2000548449A priority patent/JP2002520000A/ja
Priority to AT99924233T priority patent/ATE518956T1/de
Priority to CA002331846A priority patent/CA2331846C/fr
Publication of WO1999058658A2 publication Critical patent/WO1999058658A2/fr
Publication of WO1999058658A3 publication Critical patent/WO1999058658A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
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    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
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    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • BACKGROUND OF THE INVENTION Vaccines are of fundamental importance in modern medicine and have been highly effective in combating certain human diseases. However, despite the successful implementation of vaccination programs that have greatly limited or virtually eliminated several debilitating human diseases, there are a number of diseases that affect millions worldwide for which effective vaccines have not been developed.
  • An immune response involves two major groups of cells, lymphocytes, or white blood cells, and antigen-presenting cells.
  • the purpose of these immune response cells is to recognize foreign material, such as an infectious organism or a cancer cell, and remove that foreign material from the organism.
  • lymphocytes Two major types mediate different aspects of the immune response.
  • B cells display on their cell surface specialized proteins, called antibodies, that bind specifically to foreign material, called antigens. Effector B cells produce soluble forms of the antibody, which circulate throughout the body and function to eliminate antigen from the organism. This branch of the immune system is known as the humoral branch. Memory B cells function to recognize the antigen in future encounters by continuing to express the membrane-bound form of the antibody.
  • a second major type of lymphocyte is the T cell. T cells also have on their cell surface specialized proteins that recognize antigen but, in contrast to B cells, require that the antigen be bound to a specialized membrane protein complex, the major histocompatibility complex (MHC), on the surface of an antigen-presenting cell.
  • MHC major histocompatibility complex
  • helper T lymphocytes termed helper T lymphocytes ("HTL”) and cytotoxic T lymphocytes (“CTL”)
  • CTL cytotoxic T lymphocytes
  • the second major class of immune response cells are cells that function in antigen preservation by processing antigen for binding to MHC molecules expressed in the antigen presenting cells.
  • the processed antigen bound to MHC molecules is transferred to the surface of the cell, where the antigen-MHC complex is available to bind to T cells.
  • MHC molecules can be divided into MHC class I and class II molecules and are recognized by the two classes of T cells. Nearly all cells express MHC class I molecules, which function to present antigen to cytotoxic T lymphocytes. Cytotoxic T lymphocytes typically recognize antigen bound to MHC class I. A subset of cells called antigen-presenting cells express MHC class II molecules. Helper T lymphocytes typically recognize antigen bound to MHC class II molecules. Antigen-presenting cells include dendritic cells, macrophages, B cells, fibroblasts, glial cells, pancreatic beta cells, thymic epithelial cells, thyroid epithelial cells and vascular endothelial cells. These antigen-presenting cells generally express both MHC class I and class II molecules. Also, B cells function as both antibody-producing and antigen-presenting cells.
  • helper T lymphocyte recognizes an antigen-MHC class II complex on the surface of an antigen-presenting cell
  • the helper T lymphocyte becomes activated and produces growth factors that activate a variety of cells involved in the immune response, including B cells and cytotoxic T lymphocytes.
  • B cells For example, under the influence of growth factors expressed by activated helper T lymphocytes, a cytotoxic T lymphocyte that recognizes an antigen-MHC class I complex becomes activated.
  • CTLs monitor and eliminate cells that display antigen specifically recognized by the CTL, such as infected cells or tumor cells.
  • activation of helper T lymphocytes stimulates the activation of both the humoral and cell-mediated branches of the immune system.
  • An important aspect of the immune response is the manner in which antigen is processed so that it can be recognized by the specialized cells of the immune system. Distinct antigen processing and presentation pathways are utilized. The one is a cytosolic pathway, which results in the antigen being bound to MHC class I molecules. An alternative pathway is an endoplasmic reticulum pahtway, which bypasses the cytosol. Another is an endocytic pathway, which results in the antigen being bound to MHC class II molecules.
  • a cytosolic pathway which results in the antigen being bound to MHC class I molecules.
  • An alternative pathway is an endoplasmic reticulum pahtway, which bypasses the cytosol.
  • Another is an endocytic pathway, which results in the antigen being bound to MHC class II molecules.
  • the cytosolic pathway processes endogenous antigens that are expressed inside the cell.
  • the antigen is degraded by a specialized protease complex in the cytosol of the cell, and the resulting antigen peptides are transported into the endoplasmic reticulum, an organelle that processes cell surface molecules.
  • the antigen peptides bind to MHC class I molecules, which are then transported to the cell surface for presentation to cytotoxic T lymphocytes of the immune system.
  • Antigens that exist outside the cell are processed by the endocytic pathway.
  • antigens are taken into the cell by endocytosis, which brings the antigens into specialized vesicles called endosomes and subsequently to specialized vesicles called lysosomes, where the antigen is degraded by proteases into antigen peptides that bind to MHC class II molecules.
  • the antigen peptide-MHC class II molecule complex is then transported to the cell surface for presentation to helper T lymphocytes of the immune system.
  • an effective vaccine A variety of factors must be considered in the development of an effective vaccine. For example, the extent of activation of either the humoral or cell-mediated branch of the immune system can determine the effectiveness of a vaccine against a particular disease. Furthermore, the development of immunologic memory by inducing memory-cell formation can be important for an effective vaccine against a particular disease (Kuby, supra). For example, protection from infectious diseases caused by pathogens with short incubation periods, such as influenza virus, requires high levels of neutralizing antibody generated by the humoral branch because disease symptoms are already underway before memory cells are activated.
  • protection from infectious diseases caused by pathogens with long incubation periods does not require neutralizing antibodies at the time of infection but instead requires memory B cells that can generate neutralizing antibodies to combat the pathogen before it is able to infect target tissues. Therefore, the effectiveness of a vaccine at preventing or ameliorating the symptoms of a particular disease depends on the type of immune response generated by the vaccine.
  • Man ⁇ - traditional vaccines have relied on intact pathogens such as attenuated or inactivated viruses or bacteria to elicit an immune response.
  • these traditional vaccines have advantages and disadvantages, including reversion of an attenuated pathogen to a virulent form.
  • the problem of reversion of an attenuated vaccine has been addressed by the use of molecules of the pathogen rather than the whole pathogen.
  • immunization approaches have begun to incorporate recombinant vector vaccines and synthetic peptide vaccines (Kuby, supra).
  • DNA vaccines have also been used (Donnelly et al, Annu. Rev. Immunol. 15:617-648 (1997), which is incorporated herein by reference).
  • the use of molecules of a pathogen provides safe vaccines that circumvent the potential for reversion to a virulent form of the vaccine.
  • HIV infection is perhaps the best example where an infected host may benefit from a multispecific response. Rapid progression of HIV infection has been reported in cases where a narrowly focused CTL response is induced whereas nonprogressors tend to show a broader specificity of CTLs (Goulder et al, Nat. Med. 3:212 (1997); Borrow et al, Nat. Med. 3:205 (1997)).
  • minigene plasmid encoding the epitopes in a string-of-beads fashion.
  • Induction of CTL, HTL, and B cell responses in mice by minigene plasmids have been described by several laboratories using constructs encoding as many as 11 epitopes (An et al, J. Virol. 71 :2292 (1997); Thomson et al, J. Immunol. 157:822 (1996); Whitton et al, J. Virol. 67:348 (1993); Hanke et al, Vaccine 16:426 (1998); Vitiello et al. Eur. J. Immunol.
  • Minigenes have been delivered in vivo by infection with recombinant adenovirus or vaccinia, or by injection of purified DNA via the intramuscular or intradermal route (Thomson et al, J. Immunol. 160:1717 (1998); Toes et al, Proc. Nati. Acad. Sci. USA 94:14660 (1997)).
  • minigene DNA vaccines for human use will require addressing certain fundamental questions dealing with epitope MHC affinity, optimization of constructs for maximum in vivo immunogenicity, and development of assays for testing in vivo potency of multi-epitope minigene constructs.
  • MHC binding affinity of epitopes it is not currently known whether both high and low affinity epitopes can be included within a single minigene construct, and what ranges of peptide affinity are permissible for CTL induction in vivo. This is especially important because dominant epitopes can vary in their affinity and because it might be important to be able to deliver mixtures of dominant and subdominant epitopes that are characterized by high and low MHC binding affinities.
  • HTL helper T lymphocyte
  • CTL cytotoxic T lymphocyte
  • the invention therefore provides expression vectors encoding two or more HTL epitopes fused to a MHC class II targeting sequence, as well as expression vectors encoding a CTL epitope and a universal HTL epitope fused to an MHC class I targeting sequence.
  • the HTL epitope can be a universal HTL epitope (also referred to as a universal MHC class II epitope).
  • the invention also provides expression vectors encoding two or more HTL epitopes fused to a MHC class II targeting sequence and encoding one or more CTL epitopes.
  • the invention additionally provides methods of stimulating an immune response by administering an expression vector of the invention in vivo, as well as methods of assaying the human immunogenicity of a human T cell peptide epitope in vivo in a non-human mammal.
  • the present invention provides an expression vector comprising a promoter operably linked to a first nucleotide sequence encoding a major histocompatibility (MHC) targeting sequence fused to a second nucleotide sequence encoding two or more heterologous peptide epitopes, wherein the heterologous peptide epitopes comprise two HTL peptide epitopes or a CTL peptide epitope and a universal HTL peptide epitope.
  • MHC major histocompatibility
  • the present invention provides a method of inducing an immune response in vivo comprising administering to a mammalian subject an expression vector comprising a promoter operably linked to a first nucleotide sequence encoding a major histocompatibility (MHC) targeting sequence fused to a second nucleotide sequence encoding two or more heterologous peptide epitopes, wherein the heterologous peptide epitopes comprise two HTL peptide epitopes or a CTL peptide epitope and a universal HTL peptide epitope.
  • MHC major histocompatibility
  • the present invention provides a method of inducing an immune response in vivo comprising administering to a mammalian subject an expression vector comprising a promoter operably linked to a first nucleotide sequence encoding a major histocompatibility (MHC) targeting sequence fused to a second nucleotide sequence encoding a heterologous human HTL peptide epitope.
  • MHC major histocompatibility
  • the present invention provides a method of assaying the human immunogenicity of a human T cell peptide epitope in vivo in a non-human mammal, comprising the step of administering to the non-human mammal an expression vector comprising a promoter operably linked to a first nucleotide sequence encoding a heterologous human CTL or HTL peptide epitope.
  • the heterologous peptide epitopes comprise two or more heterologous HTL peptide epitopes.
  • the heterologous peptide epitopes comprise a CTL peptide epitope and a universal HTL peptide epitope.
  • the heterologous peptide epitopes further comprise one to two or more heterologous CTL peptide epitopes.
  • the expression vector comprises both HTL and CTL peptide epitopes.
  • one of the HTL peptide epitopes is a universal HTL epitope.
  • the universal HTL epitope is a pan DR epitope.
  • the pan DR epitope has the sequence AlaLysPheValAlaAlaTrpThrLeuLysAlaAlaAla (SEQ ID NO:38).
  • the peptide epitopes are hepatitis B virus epitopes, hepatitis C virus epitopes, human immunodeficiency virus epitopes, human papilloma virus epitopes, MAGE epitopes, PSA epitopes, PSM epitopes, PAP epitopes, p53 epitopes, CEA epitopes, Her2/neu epitopes, or Plasmodium epitopes.
  • the peptide epitopes each have a sequence selected from the group consisting of the peptides depicted in Tables 1-8. In another embodiment, at least one of the peptide epitopes is an analog of a peptide depicted in Tables 1-8.
  • the MHC targeting sequence comprises a region of a polypeptide selected from the group consisting of the Ii protein, LAMP-I, HLS-DM, HLA-DO, H2-DO, influenza matrix protein, hepatitis B surface antigen, hepatitis B virus core antigen, Ty particle, Ig- ⁇ protein, Ig- ⁇ protein, and lg kappa chain signal sequence.
  • the expression vector further comprises a second promoter sequence operably linked to a third nucleotide sequence encoding one or more heterologous HTL or CTL peptide epitopes.
  • the CTL peptide epitope comprises a stnictural motif for an HLA supertype, whereby the peptide CTL epitope binds to two or more members of the supertype with an affinity of greater that 500 nM.
  • the CTL peptide epitopes have structural motifs that provide binding affinity for more than one HLA allele supertype.
  • the non-human mammal is a transgenic mouse that expresses a human HLA allele.
  • the human HLA allele is selected from the group consisting of Al 1 and A2.1.
  • the non-human mammal is a macaque that expresses a human HLA allele.
  • Figure 1 shows the nucleotide and amino acid sequences (SEQ ID NOS:l and 2, respectively) of the IiPADRE construct encoding a fusion of the murine Ii gene with a pan DR epitope sequence substituted for the CLIP sequence of the Ii protein.
  • Figure 2 shows the nucleotide and amino acid sequences (SEQ ID NOS:3 and 4, respectively) of the I80T construct encoding a fusion of the cytoplasmic domain, the transmembrane domain and part of the luminal domain of the Ii protein fused to multiple MHC class II epitopes.
  • Figure 3 shows the nucleotide and amino acid sequences (SEQ ID NOS: 5 and 6, respectively) of the IiThfull construct encoding a fusion of the cytoplasmic domain, transmembrane domain and a portion of the luminal domain of the Ii protein fused to multiple T helper epitopes and amino acid residues 101 to 215 of the Ii protein, which encodes the trimerization region of the Ii protein.
  • Figure 4 shows the nucleotide and amino acid sequences (SEQ ID NOS:7 and 8, respectively) of the KappaLAMP-Th construct encoding a fusion of the murine immunoglobulin kappa signal sequence fused to multiple T helper epitopes and the transmembrane and cytoplasmic domains of LAMP- 1.
  • Figure 5 shows the nucleotide and amino acid sequences (SEQ ID NOS:9 and 10, respectively) of the H2M-Th construct encoding a fusion of the signal sequence of H2-M fused to multiple MHC class II epitopes and the transmembrane and cytoplasmic domains of H2-M.
  • Figure 6 shows the nucleotide and amino acid sequences (SEQ ID NOS: 11 and 12, respectively) of the H20-Th construct encoding a fusion of the signal sequence of H2-DO fused to multiple MHC class JJ epitopes and the transmembrane and cytoplasmic domains of H2-DO.
  • Figure 7 shows the nucleotide and amino acid sequences (SEQ ID NOS: 13 and 14, respectively) of the PADRE-Influenza matrix construct encoding a fusion of a pan DR epitope sequence fused to the amino-terminus of influenza matrix protein.
  • Figure 8 shows the nucleotide and amino acid sequences (SEQ ID NOS: 15 and 16, respectively) of the PADRE -HBV-s construct encoding a fusion of a pan DR epitope sequence fused to the amino-terminus of hepatitis B virus surface antigen.
  • Figure 9 shows the nucleotide and amino acid sequences (SEQ ID NOS: 17 and 18, respectively) of the Ig-alphaTh construct encoding a fusion of the signal sequence of the Ig- ⁇ protein fused to multiple MHC class II epitopes and the transmembrane and cytoplasmic domains of the Ig- ⁇ protein.
  • Figure 10 shows the nucleotide and amino acid sequences (SEQ LD
  • Ig-betaTh construct encoding a fusion of the signal sequence of the Ig- ⁇ protein fused to multiple MHC class II epitopes and the transmembrane and cytoplasmic domains of the Ig- ⁇ protein.
  • Figure 11 shows the nucleotide and amino acid sequences (SEQ ID NOS:21 and 22, respectively) of the SigTh construct encoding a fusion of the signal sequence of the kappa immunoglobulin fused to multiple MHC class II epitopes.
  • Figure 12 shows the nucleotide and amino acid sequences (SEQ LD NOS:23 and 24, respectively) of human HLA-DR, the invariant chain (Ii) protein.
  • Figure 13 shows the nucleotide and amino acid sequences (SEQ ID NOS:25 and 26, respectively) of human lysosomal membrane glycoprotein- 1 (LAMP-1).
  • Figure 14 shows the nucleotide and amino acid sequences (SEQ LD NOS:27 and 28, respectively) of human HLA-DMB.
  • Figure 15 shows the nucleotide and amino acid sequences (SEQ ID NOS:27 and 28, respectively) of human HLA-DMB.
  • Figure 16 shows the nucleotide and amino acid sequences (SEQ ID NOS:31 and 32, respectively) of the human MB-1 Ig- ⁇ .
  • Figure 17 shows the nucleotide and amino acid sequences (SEQ ID NOS:33 and 34, respectively) of human Ig- ⁇ protein.
  • Figure 18 shows a schematic diagram depicting the method of generating some of the constructs encoding a MHC class II targeting sequence fused to multiple MHC class II epitopes.
  • Figure 19 shows the nucleotide sequence of the vector pEP2 (SEQ ID NO:35).
  • Figure 20 shows the nucleotide sequence of the vector pMLN.O (SEQ ID NO:
  • Figure 21 shows the nucleotide sequence of the vector pMIN.l (SEQ ID NO:36).
  • FIG. 22 Representative CTL responses in HLA- A2.1/K b -H-2 bxs mice immunized with pMin.l DNA.
  • Splenocytes from primed animals were cultured in triplicate flasks and stimulated twice in vitro with each peptide epitope. Cytotoxicity of each culture was assayed in a 51 Cr release assay against Jurkat-A2.1/K target cells in the presence (filled symbols, solid lines) or absence (open symbols, dotted lines) of peptide. Each symbol represents the response of a single culture.
  • FIG. 23 Presentation of viral epitopes to specific CTLs by Jurkat- A2.1/K b tumor cells transfected with DNA minigene. Two constructs were used for transfection, pMin.l and pMin.2-GFP. pMin.2-GFP-transfected targets cells were sorted by FACS and the population used in this experiment contained 60% fluorescent cells. CTL stimulation was measured by quantitating the amount of IFN- ⁇ release (A, B) or by lysis of 51 Cr-labeled target cells (C, D, hatched bars). CTLs were stimulated with transfected cells (A, C) or with parental Jurkat-A2.1/K b cells in the presence of 1 ⁇ g/ml peptide (B, D).
  • FIG. 24 Summary of modified minigene constructs used to address variables critical for in vivo immunogenicity. The following modifications were incorporated into the prototype pMin.l construct; A, deletion of PADRE HTL epitope; B, incorporation of the native HBV Pol 551 epitope that contains an alanine in position 9; C, deletion of the lg kappa signal sequence; and D, switching position of the HBV Env 335 and HBV Pol 455 epitopes.
  • FIG. 25 Examination of variables that may influence pMin.l immunogenicity. In vivo CTL-inducing activity of pMin.l is compared to modified constructs. For ease of comparison, the CTL response induced by each of the modified DNA minigene constructs (shaded bars) is compared separately in each of the four panels to the response induced by the prototype pMin.l construct (solid bars). The geometric mean response of CTL-positive cultures from two to five independent experiments are shown. Numbers shown with each bar indicate the number of positive cultures/total number tested for that particular epitope.
  • the ratio of positive cultures/total tested for the pMin.l group is shown in panel A and is the same for the remaining Figure panels (see Example V, Materials and Methods, in vitro CTL cultures, for the definition of a positive CTL culture).
  • Theradigm responses were obtained by immunizing animals with the lipopeptide and stimulating and testing splenocyte cultures with the HBV Core 18-27 peptide.
  • An "HTL” peptide epitopeor an "MHC II epitope” is an MHC class II restricted epitope, i.e., one that is bound by an MHC class II molecule.
  • a “CTL” peptide epitope or an "MHC I epitope” is an MHC class I restricted epitope, i.e., one that is bound by an MHC class I molecule.
  • MHC targeting sequence refers to a peptide sequence that targets a polypeptide, e.g., comprising a peptide epitope, to a cytosolic pathway (e.g., an MHC class I antigen processing pathway), en endoplasmic reticulum pathwasy, or an endocytic pathway (e.g., an MHC class II antigen processing pathway).
  • cytosolic pathway e.g., an MHC class I antigen processing pathway
  • en endoplasmic reticulum pathwasy e.g., an MHC class II antigen processing pathway.
  • endocytic pathway e.g., an MHC class II antigen processing pathway
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
  • a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature, e.g., a fusion polypeptide comprising subsequence from different polypeptides, peptide epitopes from the same polypeptide that are not naturally in an adjacent position, or repeats of a single peptide epitope.
  • the term "universal MHC class II epitope” or a “universal HTL epitope” refers to a MHC class II peptide epitope that binds to gene products of multiple MHC class II alleles.
  • the DR, DP and DQ alleles are human MHC II alleles.
  • a unique set of peptides binds to a particular gene product of a MHC class II allele.
  • a universal MHC class II epitope is able to bind to gene products of multiple MHC class II alleles.
  • a universal MHC class II epitope binds to 2 or more MHC class II alleles, generally 3 or more MHC class II alleles, and particularly 5 or more MHC class II alleles.
  • the presence of a universal MHC class II epitope in an expression vector is advantageous in that it functions to increase the number of allelic MHC class II molecules that can bind to the peptide and, consequently, the number of Helper T lymphocytes that are activated.
  • Universal MHC class II epitopes are well known in the art and include, for example, epitopes such as the “pan DR epitopes,” also referred to as “PADRE” (Alexander et al. Immunity 1:751-761 (1994); WO 95/07707, USSN 60/036,713, USSN 60/037,432, PCTUS98/01373, 09/009,953, and USSN 60/087,192 each of which is incorporated herein by reference).
  • PADRE pan DR epitopes
  • a "pan DR binding peptide" or a "PADRE” peptide of the invention is a peptide capable of binding at least about 7 different DR molecules, preferably 7 of the 12 most common DR molecules, most preferably 9 of the 12 most common DR molecules (DR1, 2w2b, 2w2a, 3, 4w4, 4wl4, 5, 7, 52a, 52b, 52c, and 53), or alternatively, 50% of a panel of DR molecules representative of greater than or equal to 75% of the human population, preferably greater than or equal to 80% of the human population.
  • Pan DR epitopes can bind to a number of DR alleles and are strongly immunogenic for T cells. For example, pan DR epitopes were found to be more effective at inducing an immune response than natural MHC class II epitopes (Alexander, supra).
  • An example of a PADRE epitope is the peptide
  • an "epitope” is a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor proteins and/or Major Histocompatibility Complex (MHC) receptors.
  • MHC Major Histocompatibility Complex
  • an epitope is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, T cell receptor or HLA molecule.
  • epitope and peptide are often used interchangeably. It is to be appreciated, however, that isolated or purified protein or peptide molecules larger than and comprising an epitope of the invention are still within the bounds of the invention.
  • high affinity with respect to HLA class I molecules is defined as binding with an IC50 (or K D ) of less than 50 nM.
  • Intermediate affinity is binding with an IC50 (or K D ) of between about 50 and about 500 nM.
  • High affinity with respect to binding to HLA class II molecules is defined as binding with an K D of less than 100 nM.
  • Intermediate affinity is binding with a K D of between about 100 and about 1000 nM.
  • Assays for determining binding are described in detail, e.g., in PCT publications WO 94/20127 and WO 94/03205. Alternatively, binding is expressed relative to a reference peptide.
  • the IC50s of the peptides tested may change somewhat. However, the binding relative to the reference peptide will not significantly change. For example, in an assay run under conditions such that the IC50 of the reference peptide increases 10-fold, the IC50 values of the test peptides will also shift approximately 10-fold. Therefore, to avoid ambiguities, the assessment of whether a peptide is a good, intermediate, weak, or negative binder is generally based on its IC50, relative to the IC50 of a standard peptide. Throughout this disclosure, results are expressed in terms of "IC50s.”
  • IC50 is the concentration of peptide in a binding assay at which 50%> inhibition of binding of a reference peptide is observed. Given the conditions in which the assays are run (i.e., limiting HLA proteins and labeled peptide concentrations), these values approximate KD values. It should be noted that IC50 values can change, often dramatically, if the assay conditions are varied, and depending on the particular reagents used (e.g., HLA preparation, etc.). For example, excessive concentrations of HLA molecules will increase the apparent measured IC50 of a given ligand.
  • identity in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithms using default program parameters or by manual alignment and visual inspection.
  • isolated or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.
  • MHC Major histocompatibility complex
  • HLA human leukocyte antigen
  • HLA supertype or family describes sets of HLA molecules grouped on the basis of shared peptide -binding specificities. HLA class I molecules that share somewhat similar binding affinity for peptides bearing certain amino acid motifs are grouped into HLA supertypes.
  • HLA superfamily, HLA supertype family, HLA family, and HLA xx-like supertype molecules are synonyms.
  • motif refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule.
  • Peptide motifs are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.
  • a "supermotif ' is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. Thus, a preferably is recognized with high or intermediate affinity (as defined herein) by two or more HLA antigens.
  • Cross-reactive binding indicates that a peptide is bound by more than one HLA molecule; a synonym is degenerate binding.
  • the term “peptide” is used interchangeably with “oligopeptide” in the present specification to designate a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the ⁇ -amino and carboxyl groups of adjacent amino acids.
  • the preferred CTL-inducing oligopeptides of the invention are 13 residues or less in length and usually consist of between about 8 and about 11 residues, preferably 9 or 10 residues.
  • the preferred HTL-inducing oligopeptides are less than about 50 residues in length and usually consist of between about 6 and about 30 residues, more usually between about 12 and 25, and often between about 15 and 20 residues.
  • An "immunogenic peptide” or “peptide epitope” is a peptide which comprises an allele-specific motif or supermotif such that the peptide will bind an HLA molecule and induce a CTL and/or HTL response.
  • immunogenic peptides of the invention are capable of binding to an appropriate HLA molecule and thereafter inducing a cytotoxic T cell response, or a helper T cell response, to the antigen from which the immunogenic peptide is derived.
  • a “protective immune response” refers to a CTL and/or an HTL response to an antigen derived from an infectious agent or a tumor antigen, which prevents or at least partially arrests disease symptoms or progression.
  • the immune response may also include an antibody response which has been facilitated by the stimulation of helper T cells.
  • the term “residue” refers to an amino acid or amino acid mimetic incorporated into an oligopeptide by an amide bond or amide bond mimetic.
  • Synthetic peptide refers to a peptide that is not naturally occurring, but is man-made using such methods as chemical synthesis or recombinant DNA technology.
  • the nomenclature used to describe peptide compounds follows the conventional practice wherein the amino group is presented to the left (the N-terminus) and the carboxyl group to the right (the C-terminus) of each amino acid residue.
  • amino acid residue positions are referred to in a peptide epitope they are numbered in an amino to carboxyl direction with position one being the position closest to the amino terminal end of the epitope, or the peptide or protein of which it may be a part.
  • each residue is generally represented by standard three letter or single letter designations.
  • the L-form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-form for those amino acids having D-forms is represented by a lower case single letter or a lower case three letter symbol.
  • Glycine has no asymmetric carbon atom and is simply referred to as "Gly" or G.
  • an expression vector is intended to refer to a nucleic acid molecule capable of expressing an antigen of interest such as a MHC class I or class II epitope in an appropriate target cell.
  • An expression vector can be, for example, a plasmid or virus, including DNA or RNA viruses.
  • the expression vector contains such a promoter element to express an antigen of interest in the appropriate cell or tissue in order to stimulate a desired immune response.
  • Cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs) are critical for immunity against infectious pathogens; such as viruses, bacteria, and protozoa; tumor cells; autoimrnunne diseases and the like.
  • the present invention provides minigenes that encode peptide epitopes which induce a CTL and/or HTL response.
  • the minigenes of the invention also include an MHC targeting sequence. A variety of minigenes encoding different epitopes can be tested for immunogenicity using an HLA transgenic mouse.
  • the epitopes are typically a combination of at least two or more HTL epitopes, or a CTL epitope plus a universal HTL epitope, and optinally include additional HT1 and/or CTL epitopes.
  • Two, three, four, five, six, seven, eight, nine, ten, twenty, thirty, forty or about fifty different epitopes, either HTL and/or CTL, can be included in the minigene, along with the MHC targeting sequence.
  • the epitopes can have different HLA restriction.
  • Epitopes to be tested include those derived from viruses such as HIV, HBV, HCV, HSV CMV, HPV, and HTLV; cancer antigens such as p53, Her2/Neu, MAGE, PSA, human papilloma virus, and CEA; parasites such as Trypanosoma,
  • HTL and CTL Epitopes are selected and tested using the methods described in published PCT applications WO 93/07421, WO 94/02353, WO 95/01000, WO 97/04451, and WO 97/05348, herein incorporated by reference.
  • HTL and CTL Epitopes are selected and tested using the methods described in published PCT applications WO 93/07421, WO 94/02353, WO 95/01000, WO 97/04451, and WO 97/05348, herein incorporated by reference.
  • the expression vectors of the invention encode one or more MHC class II and/or class I epitopes and an MHC targeting sequence.
  • Multiple MHC class II or class I epitopes present in an expression vector can be derived from the same antigen, or the MHC epitopes can be derived from different antigens.
  • an expression vector can contain one or more MHC epitopes that can be derived from two different antigens of the same virus or from two different antigens of different viruses.
  • any MHC epitope can be used in the expression vectors of the invention.
  • any single MHC epitope or a combination of the MHC epitopes shown in Tables 1 to 8 can be used in the expression vectors of the invention.
  • peptide epitopes can be selected by one of skill in the art, e.g., by using a computer to select epitopes that contain HLA allele- specific motifs or supermotifs.
  • the expression vectors of the invention can also encode one or more universal MHC class II epitopes, e.g., PADRE (see, e.g., SEQ ID NO:38 and Table 8 of TTC docket No. 018623-006221, filed May 12, 1999, USSN ).
  • Universal MHC class II epitopes can be advantageously combined with other MHC class I and class II epitopes to increase the number of cells that are activated in response to a given antigen and provide broader population coverage of MHC-reactive alleles.
  • the expression vectors of the invention can encode MHC epitopes specific for an antigen, universal MHC class II epitopes, or a combination of specific MHC epitopes and at least one universal MHC class II epitope.
  • MHC class I epitopes are generally about 5 to 15 amino acids in length, in particular about 8 to 11 amino acids in length.
  • MHC class II epitopes are generally about 10 to 25 amino acids in length, in particular about 13 to 21 amino acids in length.
  • a MHC class I or II epitope can be derived from any desired antigen of interest.
  • the antigen of interest can be a viral antigen, surface receptor, tumor antigen, oncogene, enzyme, or any pathogen, cell or molecule for which an immune response is desired.
  • Epitopes can be selected based on their ability to bind one or multiple HLA alleles, and can also be selected using the "analog" technique described below.
  • the expression vectors of the invention encode one or more MHC epitopes operably linked to a MHC targeting sequence.
  • a MHC targeting sequence enhances the immune response to an antigen, relative to delivery of antigen alone, by directing the peptide epitope to the site of MHC molecule assembly and transport to the cell surface, thereby providing an increased number of MHC molecule-peptide epitope complexes available for binding to and activation of T cells.
  • MHC class I targeting sequences are used in the present invention, e.g., those sequences that target an MHC class I epitope peptide to a cytosolic pathway or to the endoplasmic reticulum (see, e.g., Rammensee et al, Immunogenetics 41:178-228 (1995)).
  • the cytosolic pathway processes endogenous antigens that are expressed inside the cell.
  • cytosolic proteins are thought to be at least partially degraded by an endopeptidase activity of a proteasome and then transported to the endoplasmic reticulum by the TAP molecule (transporter associated with processing).
  • endoplasmic reticulum In the endoplasmic reticulum, the antigen binds to MHC class I molecules. Endoplasmic reticulum signal sequences bypass the cytosolic processing pathway and directly target endogenous antigens to the endoplasmic reticulum, where proteolytic degradation into peptide fragments occurs.
  • MHC class I targeting sequences are well known in the art, and include, e.g., signal sequences such as those from lg kappa ,tissue plasminogen activator or insulin.
  • a prefened signal peptide is the human lg kappa chain sequence.
  • Endoplasmic reticulum signal sequences can also be used to target MHC class II epitopes to the endoplasmic reticulum, the site of MHC class I molecule assembly.
  • MHC class II targeting sequences are also used in the invention, e.g., those that target a peptide to the endocytic pathway. These targeting sequences typically direct extracellular antigens to enter the endocytic pathway, which results in the antigen being transferred to the lysosomal compartment where the antigen is proteolytically cleaved into antigen peptides for binding to MHC class II molecules.
  • a sequence that directs a MHC class II epitope to the endosomes of the endocytic pathway and/or subsequently to lysosomes, where the MHC class II epitope can bind to a MHC class II molecule is a MHC class II targeting sequence.
  • group of MHC class II targeting sequences useful in the invention are lysosomal targeting sequences, which localize polypeptides to lysosomes. Since MHC class LI molecules typically bind to antigen peptides derived from proteolytic processing of endocytosed antigens in lysosomes, a lysosomal targeting sequence can function as a MHC class II targeting sequence.
  • Lysosomal targeting sequences are well known in the art and include sequences found in the lysosomal proteins LAMP-1 and LAMP-2 as described by August et al (U.S. Patent No. 5,633,234, issued May 27, 1997), which is incorporated herein by reference.
  • HLA-DM lysosomal proteins that contain lysosomal targeting sequences
  • HLA-DM is an endosomal/lysosomal protein that functions in facilitating binding of antigen peptides to MHC class II molecules. Since it is located in the lysosome, HLA-DM has a lysosomal targeting sequence that can function as a MHC class II molecule targeting sequence (Copier et al, J. Immunol. 157:1017-1027 (1996), which is incorporated herein by reference).
  • the resident lysosomal protein HLA-DO can also function as a lysosomal targeting sequence. In contrast to the above described resident lysosomal proteins
  • HLA-DO LAMP-1 and HLA-DM, which encode specific Tyr-containing motifs that target proteins to lysosomes
  • HLA-DO is targeted to lysosomes by association with HLA-DM (Liljedahl ef al, EMBO J. 15:4817-4824 (1996)), which is incorporated herein by reference. Therefore, the sequences of HLA-DO that cause association with HLA-DM and, consequently, translocation of HLA-DO to lysosomes can be used as MHC class II targeting sequences.
  • the murine homolog of HLA-DO, H2-DO can be used to derive a MHC class II targeting sequence.
  • a MHC class II epitope can be fused to HLA- DO or H2-DO and targeted to lysosomes.
  • the cytoplasmic domains of B cell receptor subunits Ig- ⁇ and Ig- ⁇ mediate antigen internalization and increase the efficiency of antigen presentation (Bonnerot et al, Immunity 3:335-347 (1995)), which is incorporated herein by reference. Therefore, the cytoplasmic domains of the Ig- ⁇ and Ig- ⁇ proteins can function as MHC class II targeting sequences that target a MHC class II epitope to the endocytic pathway for processing and binding to MHC class II molecules.
  • MHC class II targeting sequence that directs MHC class II epitopes to the endocytic pathway is a sequence that directs polypeptides to be secreted, where the polypeptide can enter the endosomal pathway.
  • MHC class II targeting sequences that direct polypeptides to be secreted mimic the normal pathway by which exogenous, extracellular antigens are processed into peptides that bind to MHC class II molecules.
  • Any signal sequence that functions to direct a polypeptide through the endoplasmic reticulum and ultimately to be secreted can function as a MHC class II targeting sequence so long as the secreted polypeptide can enter the endosomal/lysosomal pathway and be cleaved into peptides that can bind to MHC class II molecules.
  • An example of such a fusion is shown in Figure 11, where the signal sequence of kappa immunoglobulin is fused to multiple MHC class II epitopes.
  • the Ii protein binds to MHC class II molecules in the endoplasmic reticulum, where it functions to prevent peptides present in the endoplasmic reticulum from binding to the MHC class II molecules. Therefore, fusion of a MHC class II epitope to the Ii protein targets the MHC class II epitope to the endoplasmic reticulum and a MHC class II molecule.
  • the CLIP sequence of the Ii protein can be removed and replaced with a MHC class II epitope sequence so that the MHC class II epitope is directed to the endoplasmic reticulum, where the epitope binds to a MHC class II molecule.
  • antigens themselves can serve as MHC class II or I targeting sequences and can be fused to a universal MHC class II epitope to stimulate an immune response.
  • cytoplasmic viral antigens are generally processed and presented as complexes with MHC class I molecules
  • long-lived cytoplasmic proteins such as the influenza matrix protein can enter the MHC class II molecule processing pathway (Gueguen & Long, Proc. Nati Acad. Sci. USA 93:14692-14697 (1996)), which is incorporated herein by reference. Therefore, long-lived cytoplasmic proteins can function as a MHC class II targeting sequence.
  • an expression vector encoding influenza matrix protein fused to a universal MHC class II epitope can be advantageously used to target influenza antigen and the universal MHC class II epitope to the MHC class II pathway for stimulating an immune response to influenza.
  • antigens functioning as MHC class II targeting sequences include polypeptides that spontaneously form particles.
  • the polypeptides are secreted from the cell that produces them and spontaneously form particles, which are taken up into an antigen-presenting cell by endocytosis such as receptor-mediated endocytosis or are engulfed by phagocytosis.
  • the particles are proteolytically cleaved into antigen peptides after entering the endosomal/lysosomal pathway.
  • HBV-S HBV surface antigen
  • HBV core antigen Kuhr ⁇ ber et al, International Immunol. 9: 1203-1212 (1997)), which is incorporated herein by reference.
  • yeast Ty protein yeast Ty protein
  • an expression vector containing HBV-S antigen fused to a universal MHC class II epitope can be advantageously used to target HBV-S antigen and the universal MHC class II epitope to the MHC class II pathway for stimulating an immune response to HBV.
  • HLA polymorphism The large degree of HLA polymorphism is an important factor to be taken into account with the epitope-based approach to vaccine development.
  • epitope selection encompassing identification of peptides capable of binding at high or intermediate affinity to multiple HLA molecules is preferably utilized, most preferably these epitopes bind at high or intermediate affinity to two or more allele specific HLA molecules.
  • CTL-inducing peptides of interest for vaccine compositions preferably include those that have a binding affinity for class I HLA molecules of less than 500 nM.
  • HTL-inducing peptides preferably include those that have a binding affinity for class II HLA molecules of less than 1000 nM.
  • peptide binding is assessed by testing the capacity of a candidate peptide to bind to a purified HLA molecule in vitro. Peptides exhibiting high or intermediate affinity are then considered for further analysis. Selected peptides are tested on other members of the supertype family. In prefened embodiments, peptides that exhibit cross-reactive binding are then used in vaccines or in cellular screening analyses.
  • Immunogenicity corresponds to whether an immune response is elicited at all, and to the vigor of any particular response, as well as to the extent of a population in which a response is elicited. For example, a peptide might elicit an immune response in a diverse array of the population, yet in no instance produce a vigorous response. In accordance with these principles, close to 90% of high binding peptides have been found to be immunogenic, as contrasted with about 50% of the peptides which bind with intermediate affinity. Moreover, higher binding affinity peptides leads to more vigorous immunogenic responses.
  • high binding epitopes are particularly useful.
  • the relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes on bound antigens has been determined for the first time in the art by the present inventors. The correlation between binding affinity and immunogenicity was analyzed in two different experimental approaches (Sette et al, J. Immunol. 153:5586-5592 (1994)). In the first approach, the immunogenicity of potential epitopes ranging in HLA binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice.
  • HBV hepatitis B virus
  • DR restriction was associated with intermediate affinity (binding affinities in the 100-1000 nM range). In only one of 32 cases was DR restriction associated with an IC50 of 1000 nM or greater. Thus, 1000 nM can be defined as an affinity threshold associated with immunogenicity in the context of DR molecules.
  • HLA class I and class II molecules can be classified into a relatively few supertypes, each characterized by largely overlapping peptide binding repertoires, and consensus structures of the main peptide binding pockets.
  • residues comprising the B and F pockets of HLA class I molecules as described in crystallographic studies were analyzed (Guo et al, Nature 360:364 (1992); Saper et al, J. Mol. Biol. 219:277 (1991); Madden et al, Cell 75:693 (1993); Parham et al, Immunol Rev. 143:141 (1995)).
  • residues 9, 45, 63, 66, 67, 70, and 99 were considered to make up the B pocket; and the B pocket was deemed to determine the specificity for the amino acid residue in the second position of peptide ligands.
  • residues 77, 80, 81, and 116 were considered to determine the specificity of the F pocket; the F pocket was deemed to determine the specificity for the C-terminal residue of a peptide ligand bound by the HLA class I molecule.
  • MHC class II DR molecules There is a significant difference between class I and class II HLA molecules. This difference corresponds to the fact that, although a stringent size restriction and motif position relative to the binding pocket exists for peptides that bind to class I molecules, a greater degree of heterogeneity in both size and binding frame position of the motif, relative to the N and C termini of the peptide, exists for class II peptide ligands.
  • HLA class II peptide ligands This increased heterogeneity of HLA class II peptide ligands is due to the structure of the binding groove of the HLA class II molecule which, unlike its class I counterpart, is open at both ends.
  • Crystallographic analysis of HLA class II DRB*0101- peptide complexes showed that the residues occupying position 1 and position 6 of peptides complexed with DRB*0101 engage two complementary pockets on the DRBa*0101 molecules, with the PI position corresponding to the most crucial anchor residue and the deepest hydrophobic pocket (see, e.g., Madden, Ann. Rev. Immunol. 13:587 (1995)).
  • Other studies have also pointed to the P6 position as a crucial anchor residue for binding to various other DR molecules.
  • peptides of the present invention are identified by any one of several HLA class I or II -specific amino acid motifs (see, e.g., Tables I-III of USSN 09/226,775, and 09/239,043, herein incorporated by reference in their entirety). If the presence of the motif corresponds to the ability to bind several allele-specific HLA antigens it is referred to as a supermotif.
  • the allele-specific HLA molecules that bind to peptides that possess a particular amino acid supermotif are collectively referred to as an HLA "supertype.”
  • CTL and HTL responses are not directed against all possible epitopes. Rather, they are restricted to a few "immunodominant" determinants (Zinkernagel et al, Adv. Immunol. 27:5159 (1979); Bennink et al, J. Exp. Med. 168:1935-1939 (1988); Rawle et al, J. Immunol. 146:3977-3984 (1991)).
  • CTLs recognizing at least some of the highest binding affinity peptides might be functionally inactivated. Lower binding affinity peptides are preferentially recognized at these times, and may therefore be preferred in therapeutic or prophylactic anti-cancer vaccines.
  • TAA tumor associated antigens
  • T cells to dominant epitopes may have been clonally deleted, selecting subdominant epitopes may allow extant T cells to be recruited, which will then lead to a therapeutic or prophylactic response.
  • the binding of HLA molecules to subdominant epitopes is often less vigorous than to dominant ones. Accordingly, there is a need to be able to modulate the binding affinity of particular immunogenic epitopes for one or more HLA molecules, and thereby to modulate the immune response elicited by the peptide, for example to prepare analog peptides which elicit a more vigorous response. This ability would greatly enhance the usefulness of peptide-based vaccines and therapeutic agents.
  • the strategy employed utilizes the motifs or supermotifs which correlate with binding to certain HLA class I and II molecules.
  • the motifs or supermotifs are defined by having primary anchors, and in many cases secondary anchors (see Tables I-III of USSN 09/226,775).
  • Analog peptides can be created by substituting amino acids residues at primary anchor, secondary anchor, or at primary and secondary anchor positions.
  • analogs are made for peptides that already bear a motif or supermotif.
  • Preferred secondary anchor residues of supermotifs and motifs that have been defined for HLA class I and class II binding peptides are shown in Tables II and III, respectively, of USSN 09/226,775.
  • residues are defined which are deleterious to binding to allele-specific HLA molecules or members of HLA supertypes that bind to the respective motif or supermotif (see Tables II and III of USSN 09/226,775). Accordingly, removal of such residues that are detrimental to binding can be performed in accordance with the methods described therein. For example, in the case of the A3 supertype, when all peptides that have such deleterious residues are removed from the population of analyzed peptides, the incidence of cross-reactivity increases from 22% to 37% (I., Sidney et al, Hu. Immunol. 45:79 (1996)).
  • one strategy to improve the cross-reactivity of peptides within a given supermotif is simply to delete one or more of the deleterious residues present within a peptide and substitute a small "neutral" residue such as Ala (that may not influence T cell recognition of the peptide).
  • An enhanced likelihood of cross-reactivity is expected if, together with elimination of detrimental residues within a peptide, "preferred" residues associated with high affinity binding to an allele-specific HLA molecule or to multiple HLA molecules within a superfamily are inserted.
  • the analog peptide when used as a vaccine, actually elicits a CTL response to the native epitope in vivo (or, in the case of class II epitopes, a failure to elicit helper T cells that cross-react with the wild type peptides), the analog peptide may be used to immunize T cells in vitro from ' individuals of the appropriate HLA allele. Thereafter, the immunized cells' capacity to induce lysis of wild type peptide sensitized target cells is evaluated. In both class I and class II systems it will be desirable to use as targets, cells that have been either infected or transfected with the appropriate genes to establish whether endogenously produced antigen is also recognized by the relevant T cells.
  • Another embodiment of the invention is to create analogs of weak binding peptides, to thereby ensure adequate numbers of cross-reactive cellular binders.
  • Class I peptides exhibiting binding affinities of 500-50000 nM, and carrying an acceptable but suboptimal primary anchor residue at one or both positions can be "fixed” by substituting prefened anchor residues in accordance with the respective supertype. The analog peptides can then be tested for crossbinding activity.
  • Another embodiment for generating effective peptide analogs involves the substitution of residues that have an adverse impact on peptide stability or solubility in, e.g., a liquid environment. This substitution may occur at any position of the peptide epitope.
  • a cysteine (C) can be substituted out in favor of gamma- amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity.
  • the expression vectors of the invention contain at least one promoter element that is capable of expressing a transcription unit encoding the antigen of interest, for example, a MHC class I epitope or a MHC class II epitope and an MHC targeting sequence in the appropriate cells of an organism so that the antigen is expressed and targeted to the appropriate MHC molecule.
  • a promoter element that functions in a human cell is incorporated into the expression vector.
  • An example of an expression vector useful for expressing the MHC class II epitopes fused to MHC class II targeting sequences and the MHC class I epitopes described herein is the pEP2 vector described in Example IV.
  • the minigenes are comprised of two or many different epitopes (see, e.g., Tables 1-8).
  • the nucleic acid encoding the epitopes are assembled in a minigene according to standard techniques.
  • the nucleic acid sequences encoding minigene epitopes are isolated using amplification techniques with oligonucleotide primers, or are chemically synthesized. Recombinant cloning techniques can also be used when appropriate.
  • Oligonucleotide sequences are selected which either amplify (when using PCR to assemble the minigene) or encode (when using synthetic oligonucleotides to assemble the minigene) the desired epitopes.
  • Amplification techniques using primers are typically used to amplify and isolate sequences encoding the epitopes of choice from DNA or RNA (see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al, eds, 1990)).
  • Methods such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) can be used to amplify epitope nucleic acid sequences directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. Restriction endonuclease sites can be incorporated into the primers.
  • Minigenes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
  • Synthetic oligonucleotides can also be used to construct minigenes. This method is performed using a series of overlapping oligonucleotides, representing both the sense and non-sense strands of the gene. These DNA fragments are then annealed, ligated and cloned. Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al, Nucleic Acids Res. 12:6159- 6168 (1984).
  • oligonucleotides Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).
  • the epitopes of the minigene are typically subcloned into an expression vector that contains a strong promoter to direct transcription, as well as other regulatory sequences such as enhancers and polyadenylation sites.
  • Suitable promoters are well known in the art and described, e.g., in Sambrook et al. and Ausubel et al.
  • Eukaryotic expression systems for mammalian cells are well known in the art and are commercially available.
  • Such promoter elements include, for example, cytomegalovirus (CMV), Rous sarcoma virus LTR and SV40.
  • the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the minigene in host cells.
  • a typical expression cassette thus contains a promoter operably linked to the minigene and signals required for efficient polyadenylation of the transcript. Additional elements of the cassette may include enhancers and introns with functional splice donor and acceptor sites.
  • the expression cassette can also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
  • the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
  • the particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic cells may be used. Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein Bar virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE.
  • the vector pEP2 is used in the present invention.
  • Other elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences.
  • the particular antibiotic resistance gene chosen is not critical, any of the many resistance genes known in the art are suitable.
  • the prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary.
  • the invention also provides methods for stimulating an immune response by administering an expression vector of the invention to an individual.
  • Administration of an expression vector of the invention for stimulating an immune response is advantageous because the expression vectors of the invention target MHC epitopes to MHC molecules, thus increasing the number of CTL and HTL activated by the antigens encoded by the expression vector.
  • the expression vectors of the invention are screened in mouse to determine the expression vectors having optimal activity in stimulating a desired immune response. Initial studies are therefore carried out, where possible, with mouse genes of the MHC targeting sequences.
  • Methods of determining the activity of the expression vectors of the invention are well known in the art and include, for example, the uptake of 3 H-thymidine to measure T cell activation and the release of 51 Cr to measure CTL activity as described below in Examples II and III. Experiments similar to those described in Example IV are performed to determine the expression vectors having activity at stimulating an immune response. The expression vectors having activity are further tested in human. To circumvent potential adverse immunological responses to encoded mouse sequences, the expression vectors having activity are modified so that the MHC class II targeting sequences are derived from human genes. For example, substitution of the analogous regions of the human homologs of genes containing various MHC class II targeting sequences are substituted into the expression vectors of the invention. Examples of such human homologs of genes containing MHC class II targeting sequences are shown in Figures 12 to 17. Expression vectors containing human MHC class II targeting sequences, such as those described in Example I below, are tested for activity at stimulating an immune response in human.
  • the invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an expression vector of the invention.
  • Pharmaceutically acceptable carriers are well known in the art and include aqueous or non-aqueous solutions, suspensions and emulsions, including physiologically buffered saline, alcohol/aqueous solutions or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize the expression vector or increase the absorption of the expression vector.
  • physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight polypeptides, antimicrobial agents, inert gases or other stabilizers or excipients.
  • Expression vectors can additionally be complexed with other components such as peptides, polypeptides and carbohydrates. Expression vectors can also be complexed to particles or beads that can be administered to an individual, for example, using a vaccine gun.
  • a pharmaceutically acceptable carrier including a physiologically acceptable compound, depends, for example, on the route of administration of the expression vector.
  • the invention further relates to methods of administering a pharmaceutical composition comprising an expression vector of the invention to stimulate an immune response.
  • the expression vectors are administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Feigner et al. (U.S. Patent No. 5,580,859, issued December 3, 1996); Feigner (U.S. Patent No. 5,703,055, issued December 30, 1997); and Carson et al. (U.S. Patent No. 5,679,647, issued October 21, 1997), each of which is incorporated herein by reference.
  • the minigene is administered as naked nucleic acid.
  • a pharmaceutical composition comprising an expression vector of the invention can be administered to stimulate an immune response in a subject by various routes including, for example, orally, intravaginally, rectally, or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intracisternally or by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively.
  • the composition can be administered by injection, intubation or topically, the latter of which can be passive, for example, by direct application of an ointment or powder, or active, for example, using a nasal spray or inhalant.
  • An expression vector also can be administered as a topical spray, in which case one component of the composition is an appropriate propellant.
  • the pharmaceutical composition also can be incorporated, if desired, into liposomes, microspheres or other polymer matrices (Feigner et al., U.S. Patent No. 5,703,055: Gregoriadis, Liposome Technology, Vols. I to III (2nd ed. 1993), each of which is incorporated herein by reference).
  • Liposomes for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
  • the expression vectors of the invention can be delivered to the interstitial spaces of tissues of an animal body (Feigner et al, U.S. Patent Nos. 5,580,859 and 5,703,055).
  • Administration of expression vectors of the invention to muscle is a particularly effective method of administration, including intradermal and subcutaneous injections and transdermal administration.
  • Transdermal administration such as by iontophoresis, is also an effective method to deliver expression vectors of the invention to muscle.
  • Epidermal administration of expression vectors of the invention can also be employed. Epidermal administration involves mechanically or chemically irritating the outermost layer of epidermis to stimulate an immune response to the irritant (Carson et al, U.S. Patent No. 5.679,647).
  • mucosal administration Carson et al, U.S. Patent No. 5,679,647
  • the most effective method of administration includes intranasal administration of an appropriate aerosol containing the expression vector and a pharmaceutical composition.
  • Suppositories and topical preparations are also effective for delivery of expression vectors to mucosal tissues of genital, vaginal and ocular sites.
  • expression vectors can be complexed to particles and administered by a vaccine gun.
  • the dosage to be administered is dependent on the method of administration and will generally be between about 0.1 ⁇ g up to about 200 ⁇ g.
  • the dosage can be from about 0.05 ⁇ g/kg to about 50 mg/kg, in particular about 0.005-5 mg/kg.
  • An effective dose can be determined, for example, by measuring the immune response after administration of an expression vector.
  • the production of antibodies specific for the MHC class II epitopes or MHC class I epitopes encoded by the expression vector can be measured by methods well known in the art, including ELISA or other immunological assays.
  • the activation of T helper cells or a CTL response can be measured by methods well known in the art including, for example, the uptake of 3 H-thymidine to measure T cell activation and the release of 51 Cr to measure CTL activity (see Examples II and III below).
  • compositions comprising an expression vector of the invention can be administered to mammals, particularly humans, for prophylactic or therapeutic purposes.
  • diseases that can be treated or prevented using the expression vectors of the invention include infection with HBV, HCV, HIV and CMV as well as prostate cancer, renal carcinoma, cervical carcinoma, lymphoma, condyloma acuminatum and acquired immunodeficiency syndrome (ALDS).
  • the expression vectors of the invention are administered to an individual already suffering from cancer, autoimmune disease or infected with a virus. Those in the incubation phase or acute phase of the disease can be treated with expression vectors of the invention, including those expressing all universal MHC class II epitopes, separately or in conjunction with other treatments, as appropriate.
  • pharmaceutical compositions comprising expression vectors of the invention are administered to a patient in an amount sufficient to elicit an effective immune response to an antigen and to ameliorate the signs or symptoms of a disease.
  • the amount of expression vector to administer that is sufficient to ameliorate the signs or symptoms of a disease is termed a therapeutically effective dose.
  • the amount of expression vector sufficient to achieve a therapeutically effective dose will depend on the pharmaceutical composition comprising an expression vector of the invention, the manner of administration, the state and severity of the disease being treated, the weight and general state of health of the patient and the judgment of the prescribing physician.
  • This example shows construction of expression vectors containing MHC class II epitopes that can be used to target antigens to MHC class II molecules.
  • Expression vectors comprising DNA constructs were prepared using overlapping oligonucleotides, polymerase chain reaction (PCR) and standard molecular biology techniques (Dieffenbach & Dveksler, PCR Primer: A Laboratory Manual (1995); Sambrook et al, Molecular Cloning: A Laboratory Manual (2nd ed., 1989), each of which is incorporated herein by reference).
  • PCR polymerase chain reaction
  • the full length invariant chain was amplified, cloned, and sequenced and used in the construction of the three invariant chain constructs. Except where noted, the source of cDNA for all the constructs listed below was Mouse Spleen Marathon-Ready cDNAmade from Balb/c males (Clontech; Palo Alto CA). The primer pairs were the oligonucleotide
  • GCTAGCGCCGCCACCATGGATGACCAACGCGACCTC (SEQ ID NO:40), which is designated murli-F and contains an Nhel site followed by the consensus Kozak sequence and the 5' end of the Ii cDNA; and the oligonucleotide
  • GGTACCTCACAGGGTGACTTGACCCAG (SEQ ID NO:41), which is designated murli-R and contains a Kpnl site and the 3' end of the Ii coding sequence.
  • spleen cDNA and 250 nM of each primer were combined in a 100 ⁇ l reaction with 0.25 mM each dNTP and 2.5 units oiPfu polymerase in Pfu polymerase buffer containing 10 mM KC1, 10 mM (NH 4 ) 2 S0 4 , 20 mM Tris-chloride, pH 8.75, 2 mM MgSO 4 , 0.1% TRITON X-100 and 100 ⁇ g/ml bovine serum albumin (BSA).
  • Pfu polymerase buffer containing 10 mM KC1, 10 mM (NH 4 ) 2 S0 4 , 20 mM Tris-chloride, pH 8.75, 2 mM MgSO 4 , 0.1% TRITON X-100 and 100 ⁇ g/ml bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • a Perkin/Elmer 9600 PCR machine (Perkin Elmer; Foster City CA) was used and the cycling conditions were: 1 cycle of 95°C for 5 minutes, followed by 30 cycles of 95°C for 15 seconds, 52°C for 30 seconds, and 72°C for 1 minute.
  • the PCR reaction was run on a 1% agarose gel, and the 670 base pair product was cut out, purified by spinning through a Millipore Ultrafree-MC filter (Millipore; Bedford MA) and cloned into pCR-Blunt from Invitrogen (San Diego, CA). Individual clones were screened by sequencing, and a correct clone (named bli#3) was used as a template for the helper constructs.
  • DNA constructs containing pan DR epitope sequences and MHC II targeting sequences derived from the Ii protein were prepared.
  • the Ii murine protein has been previously described (Zhu & Jones, Nucleic Acids Res. 17:447-448 (1989)), which is incorporated herein by reference.
  • the IiPADRE construct contains the full length Ii sequence with PADRE precisely replacing the CLIP region.
  • the DNA construct encodes amino acids 1 through 87 of invariant chain, followed with the 13 amino acid PADRE sequence (SEQ ID NO:38) and the rest of the invariant chain DNA sequence (amino acids 101-215).
  • the construct was amplified in 2 overlapping halves that were joined to produce the final construct.
  • the two primers used to amplify the 5' half were murli-F and the oligonucleotide
  • IiPADRE-R CAGGGTCCAGGCAGCCACGAACTTGGCCACAGGTTTGGCAGA (SEQ ID NO:42), which is designated IiPADRE-R.
  • the IiPADRE-R primer includes nucleotides 303-262 of IiPADRE. The 3' half was amplified with the primer
  • GGCTGCCTGGACCCTGAAGGCTGCCGCTATGTCCATGGATAAC (SEQ ID NO:43), which is designated IiPADRE-F and includes nucleotides 288-330 of IiPADRE; and murli-R.
  • the PCR conditions were the same as described above, and the two halves were isolated by agarose gel electrophoresis as described above. Ten microliters of each PCR product was combined in a 100 ⁇ l PCR reaction with an annealing temperature of 50°C for five cycles to generate a full length template. Primers murli-F and murli-R were added and 25 more cycles carried out.
  • the full length IiPADRE product was isolated, cloned, and sequenced as described above. This construct contains the murine Ii gene with a pan DR epitope sequence substituted for the CLIP sequence of Ii ( Figure 1).
  • a DNA construct, designated I80T, containing the cytoplasmic domain, the transmembrane domain and part of the luminal domain of Ii fused to a string of multiple MHC class II epitopes was constructed ( Figure 2). Briefly, the string of multiple MHC class II epitopes was constructed with three overlapping oligonucleotides (oligos). Each oligo overlapped its neighbor by 15 nucleotides and the final MHC class II epitope string was assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR.
  • oligo 1 nucleotides 241-310, CTTCGCATGAAGCTTATCAGCCAGGCTGTGCACGCCGCTCACGCCGAAATCAA CGAAGCTGGAAGAACCC (SEQ ID NO:44); oligo 2, nucleotides 364-295,
  • GCTGCCTGGACCCTGAAG SEQ LD NO:46.
  • oligos 1 and 2 were combined in a 100 ⁇ l reaction containing Pfu polymerase.
  • a Perkin Elmer 9600 PCR machine was used and the annealing temperature used was 45° C.
  • the PCR product was gel-purified, and a second reaction containing the PCR product of oligos 1 and 2 with oligo 3 was annealed and extended for 10 cycles before gel purification of the full length product to be used as a "mega-primer.”
  • the I80T construct was made by amplifying bli#3 with murli-F and the mega-primer.
  • the cycling conditions were: 1 cycle of 95°C for 5 minutes, followed by 5 cycles of 95°C for 15 seconds, 37°C for 30 seconds, and 72°C for 1 minute.
  • Primer Help- epR was added and an additional 25 cycles were carried out with the annealing temperature raised to 47°C.
  • GGTACCTCAAGCGGCAGCCTTCAGGGTCCAGGCA (SEQ ID NO:47) conesponds to nucleotides 438-405.
  • the full length I80T product was isolated, cloned, and sequenced as above.
  • the I80T construct ( Figure 2) encodes amino acid residues 1 through 80 of
  • Ii containing the cytoplasmic domain, the transmembrane domain and part of the luminal domain, fused to a string of multiple MHC class II epitopes corresponding to: amino acid residues 323-339 of ovalbumin (IleSerGlnAlaValHisAlaAlaHisAlaGluIleAsnGluAlaGlyArg; SEQ ID NO:48); amino acid residues 128 to 141 of HBV core antigen (amino acids
  • SEQ LD NO:50 pan DR sequence designated SEQ LD NO:38.
  • This construct, designated IiThfull encodes the first 80 amino acids of invariant chain followed by the MHC class II epitope string (replacing CLIP) and the rest of the invariant chain (amino acids 101-215). Briefly, the construct was generated as two overlapping halves that were annealed and extended by PCR to yield the final product.
  • the 5 " end of IiThfull was made by amplifying I80T with murli-F (SEQ ID NO:40) and Th-Pad-R.
  • the Th-Pad-R primer AGCGGCAGCCTTCAGGGTC corresponds to nucleotides 429-411.
  • the 3' half was made by amplifying bli#3 with IiPADRE-F and murli-R (SEQ LD NO:41).
  • the IiPADRE-F primer GGCTGCCTGGACCCTGAAGGCTGCCGCTATGTCCATGGATAAC corresponds to nucleotides 402-444.
  • Each PCR product was gel purified and mixed, then denatured, annealed, and extended by five cycles of PCR.
  • Primers murli-F SEQ ID NO:
  • primer pairs IF plus 1R designated below for each specific construct, were used to amplify the specific signal sequence and contained an overlapping 15 base pair tail identical to the 5' end of the MHC class II epitope string.
  • Primer pair Th-ova-F, ATCAGCCAGGCTGTGCACGC (SEQ ID NO:53), plus Th-Pad-R (SEQ ED NO:51) were used to amplify the MHC class II epitope string.
  • a 15 base pair overlap and the specific transmembrane and cytoplasmic tail containing the targeting signals were amplified with primer pairs 2F plus 2R.
  • All three pieces of each cDNA were amplified using the following conditions: 1 cycle of 95°C for 5 minutes, followed by 30 cycles of 95°C for 15 seconds, 52°C for 30 seconds, and 72°C for 1 minute.
  • Each of the three fragments was agrose-gel purified, and the signal sequence and MHC class II string fragments were combined and joined by five cycles in a second PCR.
  • primers IF and Th-Pad-R were added for 25 additional cycles and the PCR product was gel purified.
  • This signal sequence plus MHC class II epitope string fragment was combined with the transmembrane plus cytoplasmic tail fragment for the final PCR.
  • primers IF plus 2R were added for 25 additional cycles and the product was gel purified, cloned and sequenced.
  • This construct designated kappaLAMP-Th, contains the consensus mouse immunoglobulin kappa signal sequence and was amplified from a plasmid containing full length immunoglobulin kappa as depicted in Figure 18.
  • the primer IF used was the oligonucleotide designated KappaSig-F, GCTAGCGCCGCCACCATGGGAATGCAG (SEQ ID NO:54).
  • the primer 1R used was the oligonucleotide designated Kappa-Th-R, CACAGCCTGGCTGATTCCTCTGGACCC (SEQ ID NO:55).
  • the primer 2F used was the oligonucleotide designated PAD/LAMP -F, CTGAAGGCTGCCGCTAACAACATGTTGATCCCC (SEQ ID NO:56).
  • the primer 2R used was the oligonucleotide designated LAMP-CYTOR, GGTACCCTAGATGGTCTGATAGCC (SEQ ID NO:57).
  • H2-M-Th A DNA construct containing the signal sequence of H2-M fused to the MHC class II epitope string shown in Figure 2 and the transmembrane and cytoplasmic domains of H2-M was generated ( Figure 5).
  • the mouse H2-M gene has been described previously, Peleraux et al, Immunogenetics 43:204-214 (1996)), which is incorporated herein by reference.
  • This construct was designated H2M-Th and was constructed as depicted in Figure 18.
  • the primer IF used was the oligonucleotide designated H2-Mb- 1F, GCC GCT AGC GCC GCC ACC ATG GCT GCA CTC TGG (SEQ ID NO:58).
  • the primer 1R used was the oligonucleotide designated H2-Mb-1R, CAC AGC CTG GCT GAT CCC CAT ACA GTG CAG (SEQ ID NO:59).
  • the primer 2F used was the oligonucleotide designated H2-Mb-2F, CTG AAG GCT GCC GCT AAG GTC TCT GTG TCT (SEQ ID NO:60).
  • the primer 2R used was the oligonucleotide designated H2-Mb- 2R, GCG GGT ACC CTAATG CCG TCC TTC (SEQ ID NO:61).
  • H2-DO A DNA construct containing the signal sequence of H2-DO fused to the MHC class II epitope string shown in Figure 2 and the transmembrane and cytoplasmic domains of H2-DO was generated (Figure 6).
  • the mouse H2-DO gene has been described previously (Larhammar et al, J. Biol. Chem. 260:14111-14119 (1985)), which is incorporated herein by reference (GenBank accession No. Ml 9423).
  • This construct, designated H20-Th was constructed as depicted in Figure 18.
  • the primer IF used was the oligonucleotide designated H2-Ob-lF, GCG GCT AGC GCC GCC ACC ATG GGC GCT GGG AGG (SEQ ID NO: 62).
  • the primer 1R used was the oligonucleotide designated H2-Ob-lR, TGC ACA GCC TGG CTG ATG GAA TCC AGC CTC (SEQ ID NO:63).
  • the primer 2F used was the oligonucleotide designated H2-Ob-2F, CTG AAG GCT GCC GCT ATA CTG AGT GGA GCT (SEQ ID NO:64).
  • the primer 2R used was the oligonucleotide designated H2-Ob-2R, GCC GGT ACC TCATGT GAC ATG TCC
  • a DNA construct containing a pan DR epitope sequence (SEQ ID NO:38) fused to the amino-terminus of influenza matrix protein is generated ( Figure 7).
  • This construct designated PADRE-Influenza matrix, contains the universal MHC class II epitope PADRE attached to the amino terminus of the influenza matrix coding sequence.
  • the construct is made using a long primer on the 5' end primer.
  • the 5' primer is the oligonucleotide
  • the 3' primer is the oligonucleotide TCACTTGAATCGCTGCATCTGCACCCCCAT (SEQ ID NO: 67).
  • Influenza virus from the America Type Tissue Collection is used as a source for the matrix coding region (Perdue et al. Science 279:393-396 (1998)), which is inco ⁇ orated herein by reference (GenBank accession No. AF036358).
  • a DNA construct containing a pan DR epitope sequence (SEQ ID NO: 38) fused to the amino-terminus of HBV-S antigen was generated ( Figure 8). This construct is designated PADRE -HBV-s and was generated by annealing two overlapping oligonucleotides to add PADRE onto the amino terminus of hepatitis B surface antigen
  • the primer IF used was the oligonucleotide designated lg alpha- IF, GCG GCT AGC GCC GCC ACC ATG CCA GGG GGT CTA (SEQ ID NO:70).
  • the primer IR used was the oligonucleotide designated Igalpha-IR, GCA CAG CCT GGC TGATGG CCT GGC ATC CGG (SEQ ID NO:71).
  • the primer 2F used was the oligonucleotide designated Igalpha-2F, CTG AAG GCT GCC GCT GGG ATC ATC TTG CTG (SEQ ID NO:72).
  • the primer 2R used was the oligonucleotide designated Igalpha-2R, GCG GGT ACC TCA TGG CTT TTC CAG CTG (SEQ ID NO:73).
  • Ig-betaTh was constructed as depicted in Figure 18.
  • the primer IF used was the oligonucleotide designated B29-1F (33mer) GCG GCT AGC GCC GCC ACC ATG GCC ACA CTG GTG (SEQ ID NO:74).
  • the primer IR used was the oligonucleotide designated B29-1R (30mer) CAC AGC CTG GCT GAT CGG CTC ACC TGA GAA (SEQ ID NO:75).
  • the primer 2F used was the oligonucleotide designated B292F (30mer) CTG AAG GCT GCC GCT ATT ATC TTG ATC CAG (SEQ ID NO: 76).
  • the primer 2R used was the oligonucleotide designated B29-2R (27mer), GCC GGTACC TCATTC CTG GCC TGGATG (SEQ ID NO:77).
  • a DNA construct containing the signal sequence of the kappa immunoglobulin signal sequence fused to the MHC class II epitope string shown in Figure 2 was constructed (Figure 11). This construct is designated SigTh and was generated by using the kappaLAMP-Th construct (shown in Figure 4) and amplifying with the primer pair KappaSig-F (SEQ ID NO:54) plus Help-epR (SEQ ID NO:47) to create SigTh.
  • SigTh contains the kappa immunoglobulin signal sequence fused to the T helper epitope string and terminated with a translational stop codon.
  • LAMP-Th construct similar to Figure 4, the signal sequence encoded by amino acid residues 1-19 (nucleotides 11-67) of human LAMP- 1 ( Figure 13) (GenBank accession No. J04182), which is fused to the MHC class II epitope string, is followed by the transmembrane (nucleotides 1163-1213) and cytoplasmic tail (nucleotides 1214-1258) region encoded by amino acid residues 380-416 of human LAMP-1.
  • HLA-DM-Th construct corresponding to Figure 5, the signal sequence encoded by amino acid residues 1-17 (nucleotides 1-51) of human HLA-DMB ( Figure 14) (GenBank accession No. U15085), which is fused to the MHC class II epitope string, is followed by the transmembrane (nucleotides 646-720) and cytoplasmic tail (nucleotides 721-792) region encoded by amino acid residues 216-263 of human HLA- DMB.
  • HLA-DO-Th construct corresponding to Figure 6, the signal sequence encoded by amino acid residues 1-21 (nucleotides 1-63) of human HLA-DO ( Figure 15) (GenBank accession No. L29472 J02736 N00052), which is fused to the MHC class II epitope string, is followed by the transmembrane (nucleotides 685-735) and cytoplasmic tail (nucleotides 736-819) region encoded by amino acid residues 223-273 of human HLA-DO.
  • the SigTh construct shown in Figure 11 can be used in mouse and human. Alternatively, a signal sequence derived from an appropriate human gene containing a signal sequence can be substituted for the mouse kappa immunoglobulin sequence in the Sig Th construct.
  • PADRE-HBVs construct shown in Figure 8 can be used in mouse and human.
  • the pEP2 vector was constructed to contain dual CMV promoters.
  • the pEP2 vector used the backbone of pcDNA3.1 (-)Myc-His A from Invitrogen and pLRESlhyg from Clontech. Changes were made to both vectors before the CMV transcription unit from pLRESlhyg was moved into the modified pcDN A vector.
  • the pcDNA3.1(-)Myc-His A vector http://www.invitrogen.com was modified. Briefly, the PvuII fragment (nucleotides 1342-3508) was deleted. A BspHI fragment that contains the Ampicillin resistance gene (nucleotides 4404-5412) was cut out. The Ampicillin resistance gene was replaced with the kanamycin resistance gene from pUC4K (GenBank Accession #X06404).
  • pUC4K was amplified with the primer set: TCTGATGTTACATTGCACAAG (SEQ ID NO:78) (nucleotides 1621-1601) and GCGCACTCATGATGCTCTGCCAGTGTTACAACC (SEQ ID NO:79) (nucleotides 682-702 plus the addition of a BspHI restriction site on the 5' end).
  • the PCR product was digested with BspHI and ligated into the vector digested with BspHI. The region between the Pmel site at nucleotide 905 and the EcoRV site at nucleotide 947 was deleted.
  • the vector was then digested with Pmel (cuts at nucleotide 1076) and Apal (cuts at nucleotide 1004), Klenow filled in at the cohesive ends and ligated.
  • the Kpnl site at nucleotide 994 was deleted by digesting with Kpnl and filling in the ends with Klenow DNA polymerase, and ligating.
  • the intron A sequence from CMV was added by amplifying CMV DNA with the primer set: GCGTCTAGAGTAAGTACCGCCTATAGACTC (SEQ ID NO: 80) (nucleotides 635-655 plus an Xbal site on the 5' end) and CCGGCTAGCCTGCAGAAAAGACCCATGGAA (SEQ ID NO:81) (nucleotides 1461-1441 plus an Nhel site on the 3' end).
  • the PCR product was digested with Xbal and Nhel and ligated into the Nhel site of the vector
  • the Kpnl site (nucleotide 911) was deleted by cutting and filling in with Klenow.
  • the plasmid was cut with Notl (nucleotide 1254) and Xbal (nucleotide 3196) and a polylinker oligo was inserted into the site.
  • the polylinker was formed by annealing the following two oligos:
  • CTAGCGGTACCGGCATTAGTCTATGGCCCGACTCTAGATTTTTTCCTTGC (SEQ ID NO:83).
  • the resulting plasmid was cut with Hindi and the fragment between Hindi sites 234 and 3538 was isolated and ligated into the modified pcDNA vector. This fragment contains a CMV promoter, intron, polylinker, and polyadenylation signal.
  • the pLREShyg piece and the pcDNA piece were combined to form pEP2.
  • the modified pcDNA3.1(-)Myc-His A vector was partially digested with PvuII to isolate a linear fragment with the cut downstream of the pcDNA polyadenylation signal (the other PvuII site is the CMV intron).
  • the Hindi fragment from the modified pLRESlhyg vector was ligated into the PvuII cut vector.
  • the polyadenylation signal from the pcDNA derived transcription unit was deleted by digesting with EcoRI (pcDNA nucleotide 955) and Xhol (pLRESlhyg nucleotide 3472) and replaced with a synthetic polyadenylation sequence.
  • the synthetic polyadenylation signal was described in Levitt et al, Genes and Development 3:1019-1025 (1989)).
  • oligos Two oligos were annealed to produce a fragment that contained a polylinker and polyadenylation signal with EcoRI and Xhol cohesive ends.
  • the oligos were:
  • the resulting vector is named pEP2 and contains two separate transcription units. Both transcription units use the same CMV promoter but each contains different intron, polylinker, and polyadenylation sequences.
  • the pEP2 vector contains two transcription units.
  • the first transcription unit contains the CMV promoter initially from pcDNA (nucleotides 210-862 in Figure 19), CMV intron A sequence (nucleotides 900-1728 in Figure 19), polylinker cloning site (nucleotides 1740-1760 in Figure 19) and synthetic polyadenylation signal (nucleotides 1764-1769 in Figure 19).
  • the second transcription unit which was initially derived from pLRESlhyg, contains the CMV promoter (nucleotides 3165-2493 in Figure 19), intron sequence (nucleotides 2464-2173 in Figure 19), polylinker clone site (nucleotides 2126- 2095 in Figure 19) and bovine growth hormone polyadenylation signal (nucleotides 1979- 1974 in Figure 19).
  • the kanamycin resistance gene is encoded in nucleotides 4965-4061 ( Figure 19).
  • the DNA constructs described above were digested with Nhel and Kpnl and cloned into the Xbal and Kpnl sites of pEP2 (the second transcription unit).
  • AOS that contains nine MHC class I epitopes.
  • the AOS insert was initially constructed in the vector pMIN.O ( Figure 20; SEQ ID NO:36). Briefly, the AOS insert contains nine MHC class I epitopes, six restricted by HLA-A2 and three restricted by HLA-A11, and the universal MHC class II epitope PADRE.
  • the vector pMIN.O contains epitopes from HBV, HIV and a mouse ovalbumin epitope.
  • the MHC class I epitopes appear in pMIN.O in the following order: consensus mouse lg Kappa signal sequence (pMIN.O amino acid residues).
  • nucleotides 16-81) MQVQIQSLFLLLLWVPGSRG (SEQ ID NO:86) encoded by nucleotides ATG CAG GTG CAG ATC CAG AGC CTG TTT CTG CTC CTC CTG TGG GTG CCC GGG TCC AGA GGA (SEQ ID NO:87); HBV pol 149-159 (All restricted) (pMIN.O amino acid residues 21-31, nucleotides 82-114)
  • HTLWKAGILYK (SEQ ID NO:88) encoded by nucleotides CAC ACC CTG TGG AAG GCC GGA ATC CTG TAT AAG (SEQ ID NO:89);
  • PADRE-universal MHC class II epitope (pMLN.O amino acid residues 32- 45, nucleotides 115-153) AKFVAAWTLKAAA (SEQ ID NO:38) encoded by nucleotides GCCAAGTTC GTG GCT GCC TGGACC CTGAAG GCT GCC GCT (SEQ ID NO:90);
  • HBV core 18-27 (A2 restricted) (pMIN.O amino acid residues 46-55, nucleotides 154-183) FLPSDFFPSV (SEQ LD NO:91) encoded by nucleotides TTC CTG CCT AGC GAT TTC TTT CCT AGC GTG (SEQ LD NO:92); HIV env 120-128 (A2 restricted) (pMIN.O amino acid residues 56-64, nucleotides 184-210) KLTPLCVTL (SEQ LD NO:93) encoded by nucleotides AAG CTG ACC CCA CTG TGC GTG ACC CTG (SEQ ID NO:94); HBV pol 551-559 (A2 restricted) (pMIN.O amino acid residues 65-73, nucleotides 211-237) YMDDVVLGA (SEQ ID NO:95) encoded by nucleotides TAT ATG GAT GAC GTG GTG CTG GGA GCC (SEQ ID NO:96); mouse ovalbumin
  • HBV pol 455-463 (A2 restricted) (pMIN.O amino acid residues 82-90, nucleotides 262-288) GLSRYVARL (SEQ ID NO:99) encoded by nucleotides GGA CTG TCC AGATAC GTG GCT AGG CTG (SEQ ID NO: 100);
  • HTV pol 476-84 (A2 restricted) (pMIN.O amino acid residues 91-99, nucleotides 289-315)
  • ILKEPVHGV (SEQ ID NO: 101) encoded by nucleotides ATC CTG AAG GAG CCT GTG CAC GGC GTG (SEQ ID NO: 102);
  • STLPETTVVRR (SEQ ID NO: 103) encoded by nucleotides TCC ACC CTG CCA GAG ACC ACC GTG GTG AGG AGA (SEQ ID NO: 104);
  • HIV env 49-58 (All restricted) (pMIN.O amino acid residues 111-120, nucleotides 349-378) TVYYGVPVWK (SEQ ID NO: 105) encoded by nucleotides ACC GTG TAC TAT GGA GTG CCT GTG TGG AAG (SEQ ID NO: 106); and HBV env 335-343 (A2 restricted) (pMIN.O amino acid residues 121-129, nucleotides 378-405) WLSLLVPFV (SEQ LD NO: 107) encoded by nucleotides TGG CTG AGC CTG CTG GTG CCC TTT GTG (SEQ ID NO: 108).
  • the pMIN.O vector contains a Kpnl restriction site (pMIN.O nucleotides 406-411) and a Nhel restriction site (pMIN.O nucleotides 1-6).
  • the pMIN.O vector contains a consensus Kozak sequence (nucleotides 7-18) (GCCGCCACCATG; SEQ ID NO: 109) and murine Kappa Ig-light chain signal sequence followed by a string of 10 MHC class I epitopes and one universal MHC class II epitope.
  • the pMIN.O sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
  • the pMIN.O vector was constructed with eight oligonucleotides:
  • Min8 oligo GTGGAAGTGGCTGAGCCTGCTGGTGCCCTTTGTGGGTACCTGATCTAGAGC (SEQ ID NO: 117).
  • flanking primer 5' GCG CAA GAA GCT TGC TAG CG (SEQ ID NO: 118) and flanking primer 3', GCT CTA GAT CAG GTA CCC CAC (SEQ ID N0:119).
  • the original pMIN.O minigene construction was carried out using eight overlapping oligos averaging approximately 70 nucleotides in length, which were synthesized and HPLC purified by Operon Technologies Inc. Each oligo overlapped its neighbor by 15 nucleotides, and the final multi-epitope minigene was assembled by extending the overlapping oligos in three sets of reactions using PCR (Ho et al, Gene 77:51-59 (1989).
  • the full length dimer products were gel-purified, and two reactions containing the product of 1-2 and 3-4, and the product of 5-6 and 7-8 were mixed, annealed and extended for 10 cycles. Half of the two reactions were then mixed, and 5 cycles of annealing and extension carried out before flanking primers were added to amplify the full length product for 25 additional cycles.
  • the full length product was gel purified and cloned into pCR-blunt (Invitrogen) and individual clones were screened by sequencing.
  • the Min insert was isolated as an Nhel-Kpnl fragment and cloned into the same sites of pcDNA3.1 (-)/Myc-His A (Invitrogen) for expression.
  • the Min protein contains the Myc and His antibody epitope tags at its carboxyl-terminal end.
  • pMIN.O Three changes to pMIN.O were made to produce pMIN.l ( Figure 21; SEQ ID N0:37, also referred to as pMIN-AOS).
  • the mouse ova epitope was removed, the position 9 alanine anchor residue (#547) of HBV pol 551-560 was converted to a valine which increased the in vitro binding affinity 40-fold, and a translational stop codon was introduced at the end of the multi-epitope coding sequence.
  • the changes were made by amplifying two overlapping fragments and combining them to yield the full length product.
  • the first reaction used the 5' pcDNA vector primer T7 and the primer Min- ovaR (nucleotides 247-218) TGGACAGTCCCACTCCCAGCACCACGTCAT (SEQ ID NO:120).
  • Min-ovaF nucleotides 228-257
  • GCTGGGAGTGGGACTGTCCAGGTACGTGGC SEQ ID NO: 121
  • Min-StopR nucleotides 390-361 GGTACCTCACACAAAGGGCACCAGCAGGC (SEQ ID NO: 122)
  • the two fragments were gel purified, mixed, denatured, annealed, and filled in with five cycles of PCR.
  • the full length fragment was amplified with the flanking primers T7 and Min-Stop for 25 more cycles.
  • the product was gel purified, digested with Nhel and Kpnl and cloned into pcDNA3.1 for sequencing and expression.
  • the insert from pMin.1 was isolated as an Nhel-Kpnl fragment and cloned into pEP2 to make pEP2- AOS.
  • EXAMPLE II Assay for T Helper Cell Activation
  • This example shows methods for assaying T helper cell activity.
  • One method for assaying T helper cell activity uses spleen cells of an immunized organism. Briefly, a spleen cell pellet is suspended with 2-3 ml of red blood cell lysis buffer containing 8.3 g liter ammonium chloride in 0.001 M Tris-HCl, pH 7.5. The cells are incubated in lysis buffer for 3-5 min at room temperature with occasional vortexing. An excess volume of 50 ml of RIO medium is added to the cells, and the cells are pelleted. The cells are resuspended and pelleted one or two more times in R2 medium or RIO medium.
  • the cell pellet is suspended in RIO medium and counted. If the cell suspension is aggregated, the aggregates are removed by filtration or by allowing the aggregates to settle by gravity.
  • the cell concentration is brought to 10 7 /ml, and 100 ⁇ l of spleen cells are added to 96 well flat bottom plates. Dilutions of the appropriate peptide, such as pan DR epitope (SEQ ID NO: 1
  • N0:145 are prepared in R10 medium at 100, 10, 1, 0.1 and 0.01 ⁇ g/ml, and 100 ⁇ l of peptide are added to duplicate or triplicate wells of spleen cells. The final peptide concentration is 50, 5, 0.5, 0.05 and 0.005 ⁇ g/ml. Control wells receive 100 ⁇ l R10 medium. The plates are incubated for 3 days at 37°C. After 3 days, 20 ⁇ l of
  • H-thymidine 50 ⁇ Ci/ml 3 H-thymidine is added per well. Cells are incubated for 18-24 hours and then harvested onto glass fiber filters. The inco ⁇ oration of H-thymidine into DNA of proliferating cells is measured in a beta counter.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • recombinant interleukin-2 (IL-2) is added at a final concentration of 10 ng/ml. Cultures are fed every 3 days by aspirating 1 ml of medium and replacing with fresh medium containing IL-2. Two additional stimulations of the T cells with antigen are performed on approximately days 14 and 28.
  • the T cells (3 x 10 5 /well) are stimulated with peptide (10 ⁇ g/ml) using autologous PBMC cells (2 x 10 6 irradiated cells/well) (irradiated with 7500 rads) as antigen-presenting cells in a total of three wells of a 24 well tissue culture plate.
  • T cell proliferativ ⁇ responses are determined under the following conditions: 2 x 10 4 T cells/well; 1 x 10 5 irradiated PBMC/well as antigen-presenting cells; peptide concentration varying between 0.01 and 10 ⁇ g/ml final concentration.
  • the proliferation of the T cells is measured 3 days later by the addition of H-thymidine (1 ⁇ Ci/well) 18 hr prior to harvesting the cells. Cells are harvested onto glass filters and 3 H-thymidine inco ⁇ oration is measured in a beta plate counter.
  • This example shows a method for assaying cytotoxic T lymphocyte (CTL) activity.
  • CTL cytotoxic T lymphocyte
  • a CTL response is measured essentially as described previously (Vitiello et al, Eur. J. Immunol. 27:671-678 (1997), which is inco ⁇ orated herein by reference). Briefly, after approximately 10-35 days following DNA immunization, splenocytes from an animal are isolated and co-cultured at 37°C with syngeneic, irradiated (3000 rad) peptide- coated LPS blasts (1 x 10 6 to 1.5 x 10 6 cells/ml) in 10 ml R10 in T25 flasks.
  • LPS blasts are obtained by activating splenocytes (1 x 10 6 to 1.5 x 10 6 cells/ml) with 25 ⁇ g/ml lipopolysaccharides (LPS) (Sigma cat. no. L-2387; St. Louis, MO) and 7 ⁇ g/ml dextran sulfate (Pharmacia Biotech) in 30 ml R10 medium in T75 flasks for 3 days at 37°C.
  • LPS lipopolysaccharides
  • lymphoblasts are then resuspended at a concentration of 2.5 x 10 to 3.0 x 10 /ml, irradiated (3000 rad). and coated with the appropriate peptides (lOO ⁇ g/ml) for 1 h at 37°C.
  • Cells are washed once, resuspended in R10 medium at the desired concentration and added to the responder cell preparation. Cultures are assayed for cytolytic activity on day 7 in a 51 Cr-release assay.
  • target cells are labeled for 90 min at 37°C with 150 ⁇ l sodium 51 chromate ( 51 Cr) (New England Nuclear; Wilmington DE), washed three times and resuspended at the appropriate concentration in R10 medium.
  • 51 Cr sodium 51 chromate
  • 10 4 target cells are incubated in the presence of different concentrations of effector cells in a final volume of 200 ⁇ l in U-bottom 96 well plates in the presence or absence of 10 ⁇ g/ml peptide.
  • tne percent release data is transformed to lytic units 30 per 10 6 cells (LU30/10 6 ), with 1 LU30 defined as the number of effector cells required to induce 30% lysis of 10 4 target cells in a 6 h assay.
  • LU values represent the LU30/10 6 obtained in the presence of peptide minus LU30/10 6 in the absence of peptide.
  • EXAMPLE IV T Cell Proliferation in Mice Immunized with Expression Vectors Encoding MHC Class II Epitopes and MHC Class II Targeting Sequences This example demonstrates that expression vectors encoding MHC class II epitopes and MHC class II targeting sequences are effective at activating T cells.
  • the constructs used in the T cell proliferation assay are described in Example I and were cloned into the vector pEP2, a CMV driven expression vector.
  • the peptides used for T cell in vitro stimulation are: Ova 323-339, ISQAVHAAHAEINEAGR (SEQ ID NO: 123); HBVcorel28, TPPAYRPPNAPILF (SEQ ID NO: 124); HBVenvl82, FFLLTRILTLPQSLD (SEQ ID NO: 125); and PADRE, AKFVAAWTLKAAA (SEQ ID NO:38).
  • T cell proliferation was assayed essentially as described in Example II. Briefly, 12 to 16 week old B6D2 FI mice (2 mice per construct) were injected with 100 ⁇ g of the indicated expression vector (50 ⁇ g per leg) in the anterior tibialis muscle. After eleven days, spleens were collected from the mice and separated into a single cell suspension by Dounce homogenization. The splenocytes were counted and one million splenocytes were plated per well in a 96-well plate. Each sample was done in triplicate. Ten ⁇ g/ml of the corresponding peptide encoded by the respective expression vectors was added to each well. One well contained splenocytes without peptide added for a negative control. Cells were cultured at 37°C, 5% C0 2 for three days.
  • T cell proliferation assay is presented as the stimulation index (SI); this is defined as the ratio of the average 3 H-thymidine inco ⁇ oration in the presence of antigen divided by the 3 H-thymidine inco ⁇ oration in the absence of antigen.
  • SI stimulation index
  • the immunogen "PADRE + LFA” is a positive control where the PADRE peptide in incomplete Freund's adjuvant was injected into the mice and compared to the response seen by injecting the MHC class II epitope constructs containing a PADRE sequence. As shown in Table 9, most of the expression vectors tested were effective at activating T cell proliferation in response to the addition of PADRE peptide. The activity of several of the expression vectors was comparable to that seen with immunization with the PADRE peptide in incomplete Freund's adjuvant. The expression vectors containing both MHC class I and MHC class II epitopes, pEP2-AOS and pcDNA-AOS, were also effective at activating T cell proliferation in response to the addition of PADRE peptide.
  • EXAMPLE V In vivo assay Using Transgenic Mice A. Materials and methods
  • Peptides were synthesized according to standard F-moc solid phase synthesis methods which have been previously described (Ruppert et al, Cell 74:929 (1993); Sette et al, Mol. Immunol 31:813 (1994)). Peptide purity was determined by analytical reverse-phase HPLC and purity was routinely >95%. Synthesis and purification of the Theradigm-HBV lipopeptide vaccine is described in (Vitiello et al, J. Clin. Invest. 95:341 (1995)).
  • HLA-A2.1 transgenic mice used in this study were the FI generation derived by crossing transgenic mice expressing a chimeric gene consisting of the ⁇ l, ⁇ 2 domains of HLA-A2.1 and ⁇ 3 domain of H-2K b with SJL/J mice (Jackson Laboratory, Bar Harbor, ME). This strain will be referred to hereafter as HLA-A2.1/K b -H-2 bxs .
  • the parental HLA-A2.1 K b transgenic strain was generated on a C57BL/6 background using the transgene and methods described in (Vitiello et al, J. Exp. Med. 173:1007 (1991)).
  • HLA-A11/K b transgenic mice used in the current study were identical to those described in (Alexander et al, J. Immunol. 159:4753 (1997)).
  • Cell lines, MHC purification, and peptide binding assay Target cells for peptide-specific cytotoxicity assays were Jurkat cells transfected with the HLA-A2.1/K b chimeric gene (Vitiello et al, J. Exp. Med. 173:1007 (1991)) and .221 tumor cells transfected with HLA-A11/K b (Alexander et al, J. Immunol 159:4753 (1997)).
  • Jurkat- A2.1/K b cells were transfected with the pMin.l or pMin.2-GFP minigenes then tested in a cytotoxicity assay against epitope-specific CTL lines.
  • Jurkat- A2.1/K b cells were resuspended at 10 7 cells/ml and 30 ⁇ g of DNA was added to 600 ⁇ l of cell suspension. After electroporating cells in a 0.4 cm cuvette at 0.25 kV, 960 ⁇ Fd, cells were incubated on ice for 10 min then cultured for 2 d in RPMI culture medium.
  • GFP green fluorescent protein
  • CM culture medium
  • RPMI 1640 medium with Hepes (Life Technologies, Grand Island, NY) supplemented with 10% FBS, 4 mM L-glutamine, 5 X 10 "5 M 2-ME, 0.5 mM sodium pyruvate, 100 ⁇ g/ml streptomycin, and 100 U/ml penicillin.
  • pMIN.O and pMIN.l i.e., pMIN-AOS were constructed as described above and in USSN 60/085,751.
  • pMin.l -No PADRE and pMin.l -Anchor. pMin.l was amplified using two overlapping fragments which was then combined to yield the full length product.
  • the first reaction used the 5 ' pcDNA vector primer T7 and either primer ATCGCTAGGCAGGAACTTATACAGGATTCC (SEQ LD NO:126) for pMin.l-No PADRE or TGGACAGTCCGGCTCCCAGCACCACGT (SEQ LD NO:127) for pMin.l- Anchor.
  • the 3 ' half was amplified with the primers TTCCTGCCTAGCGATTTC (SEQ JD NO: 128) (No PADRE) or GCTGGGAGCCGGACTGTCCAGGTACGT (SEQ ID NO:129) (Anchor) and Min-StopR.
  • the two fragments generated from amplifying the 5' and 3 ' ends were gel purified, mixed, denatured, annealed, and filled in with five cycles of PCR.
  • the full length fragment was furtner amplified with the flanking primers T7 and Min-StopR for 25 more cycles.
  • the lg signal sequence was deleted from pMin.l by PCR amplification with primer GCTAGCGCCGCCACCATGCACACCCTGTGGAAGGC CGGAATC (SEQ LD NO: 130) and pcDNA rev (Invitrogen) primers. The product was cloned into pCR-blunt and sequenced.
  • fragment 1 was combined with the product of 2 and 3, and extended, gel purified and cloned into pcDNA3.1 for expression.
  • pMin.2-GFP The signal sequence was deleted from pMin.O by PCR amplification with Min.O-No Sig-5' plus pcDNA rev (Invitrogen) primers GCTAGCGCCGCCACCATGCACACCCTGTGGAAGGCCGGAATC (SEQ ID NO:135). The product was cloned into pCR-blunt and sequenced. The insert containing the open reading frame of the signal sequence-deleted multi-epitope construct was cut out with Nhel plus HindUl and ligated into the same sites of pEGFPNl (Clontech). This construct fuses the coding region of the signal-deleted pMin.O construct to the N-terminus of green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • mice were pretreated by injecting 50 ⁇ l of 10 ⁇ M cardiotoxin (Sigma Chem. Co., #C9759) bilaterally into the tibialis anterior muscle. Four or five days later, 100 ⁇ g of DNA diluted in PBS were injected in the same muscle.
  • Theradigm-HBV lipopeptide (10 mg/ml in DMSO) that was stored at - 20°C, was thawed for 10 min at 45°C before being diluted 1 :10 (v/v) with room temperature PBS.
  • the lipopeptide suspension was vortexed vigorously and 100 ⁇ l was injected s.c. at the tail base (100 ⁇ g/mouse).
  • Immunogenicity of individual CTL epitopes was tested by mixing each CTL epitope (50 ⁇ g/mouse) with the HBV core 128-140 peptide (TPPAYRPPNAPIL (SEQ ID NO: 124), 140 ⁇ g/mouse) which served to induce I-A b -restricted Th cells.
  • the peptide cocktail was then emuslifed in incomplete Freund's adjuvant (Sigma Chem. Co.) and 100 ⁇ l of peptide emulsion was injected s.c. at the tail base.
  • Splenocytes from cDNA-primed animals were stimulated in vitro with each of the peptide epitopes represented in the minigene.
  • Splenocytes (2.5-3.0 X 10 7 /flask) were cultured in upright 25 cm 2 flasks in the presence of 10 ⁇ g/ml peptide and 10 7 irradiated spleen cells that had been activated for 3 days with LPS (25 ⁇ g/ml) and dextran sulfate (7 ⁇ g/ml).
  • Triplicate cultures were stimulated with each epitope. Five days later, cultures were fed with fresh CM.
  • CTLs After 10 d of in vitro culture, 2-4 X 10 6 CTLs from each flask were restimulated with 10 7 LPS/dextran sulfate- activated splenocytes treated with 100 ⁇ g/ml peptide for 60-75 min at 37°C, then irradiated 3500 rads. CTLs were restimulated in 6-well plates in 8 ml of cytokine-free CM. Eighteen hr later, cultures received cytokines contained in con A-activated splenocyte supernatant (10-15% final concentration, v/v) and were fed or expanded on the third day with CM containing 10-15% cytokine supernate.
  • CTL activity of each culture was measured by incubating varying numbers of CTLs with 10 4 51 Cr-labelled target cells in the presence or absence of peptide.
  • YAC-1 cells ATCC
  • YAC- 1 : 51 Cr-labeled target cell ratio 20:1.
  • CTL activity against the HBV Pol 551 epitope was measured by stimulating DNA-primed splenocytes in vitro with the native A- containing peptide and testing for cytotoxic activity against the same peptide.
  • E:T ratio vs % cytotoxicity data curves were converted into LU per 10 6 effector cells with one LU defined as the lytic activity required to achieve 30% lysis of target cells at a 100:1 E:T ratio.
  • Specific CTL activity ( ⁇ LU) was calculated by subtracting the LU value obtained in the absence of peptide from the LU value obtained with peptide.
  • a given culture was scored positive for CTL induction if all of the following criteria were met: 1) ⁇ LU >2; 2) LU(+ peptide) ⁇ LU(- peptide) > 3; and 3) a >10% difference in % cytotoxicity tested with and without peptide at the two highest E:T ratios (starting E:T ratios were routinely between 25-50:1).
  • CTL lines were generated from pMin.l -primed splenocytes through repeated weekly stimulations of CTLs with peptide-treated LPS/DxS -activated splenocytes using the 6-well culture conditions described above with the exception that CTLs were expanded in cytokine-containing CM as necessary during the seven day stimulation period.
  • HBV Pol 551 was studied in two alternative forms: either the wild type sequence or an analog (HBV Pol 551-V) engineered for higher binding affinity.
  • the immunogenicity of the six A2.1- and three Al 1 -restricted CTL epitopes in transgenic mice was verified by co-immunization with a helper T cell peptide in an IFA formulation. All of the epitopes induced significant CTL responses in the 5 to 73 ⁇ LU range (Table 10). As mentioned above, to improve the MHC binding and immunogenicity of HBV Pol 551, the C-terminal A residue of this epitope was substituted with V resulting in a dramatic 40- fold increase in binding affinity to HLA-A2.1 (Table 10). While the parental sequence was weakly or nonimmunogenic in HLA transgenic mice, the HBV Pol 551-V analog induced significant levels of CTL activity when administered in IFA (Table 10).
  • V analog of the HBV Pol 551 epitope was selected for the initial minigene construct.
  • CTL responses were measured with target cells coated with the native HBV Pol 551 epitope, irrespective of whether the V analog or native epitope was utilized for immunization.
  • PADRE Th cell epitope PADRE was also inco ⁇ orated into the minigene.
  • PADRE has been shown previously to have high MHC binding affinity to a wide range of mouse and human MHC class II haplotypes (Alexander et al, Immunity 1 :751 (1994)).
  • PADRE is highly immunogenic in H-2 b mice that are used in the current study (Alexander et al, Immunity 1:751 (1994)).
  • pMin.l the prototype cDNA minigene construct encoding nine CTL epitopes and PADRE, was synthesized and subcloned into the pcDNA3.1 vector. The position of each of the nine epitopes in the minigene was optimized to avoid junctional mouse H-2 b and HLA-A2.1 class I MHC epitopes. The mouse lg K signal sequence was also included at the 5' end of the construct to facilitate processing of the CTL epitopes in the endoplasmic reticulum (ER) as reported by others (Anderson et al, J. Exp. Med. 174:489 (1991)).
  • ER endoplasmic reticulum
  • an ATG stop codon was introduced at the 3' end of the minigene construct upstream of the coding region for c- myc and poly-his epitopes in the pcDNA3.1 vector.
  • HLA-A2.1/K b -H-2 bxs transgenic mice were immunized intramuscularly with 100 ⁇ g of naked cDNA.
  • a control group of animals was also immunized with Theradigm-HBV, a palmitolyated lipopeptide consisting of the HBV Core 18 CTL epitope linked to the tetanus toxin 830-843 Th cell epitope.
  • Splenocytes from immunized animals were stimulated twice with each of the peptide epitopes encoded in the minigene, then assayed for peptide-specific cytotoxic activity in a 51 Cr release assay.
  • the majority of the cultures stimulated with the different epitopes exceeded 50% specific lysis of target cells at an E:T ratio of 1 : 1.
  • the % cytotoxicity values for each splenocyte culture was converted to ⁇ LU and the mean ⁇ LU of CTL activity in positive cultures for each epitope was determined (see Example V, materials and methods, for positive criteria).
  • Theradigm-HBV lipopeptide a vaccine formulation known for its high CTL-inducing potency (Vitiello et al, J. Clin. Invest. 95:341 (1995); Livingston et al, J. Immunol. 159:1383 (1997)).
  • the HBV Env 335 epitope was the only epitope showing a lower mean ALU response compared to lipopeptide (Table 11, 44 vs 349 ⁇ LU).
  • the decreased CTL response observed against HBV Env 335 was somewhat unexpected since this epitope had good A2.1 binding affinity (IC50%, 5 nM) and was also immunogenic when administered in IFA.
  • the lower response may be due, at least in part, to the inefficient processing of this epitope from the minigene polypeptide by antigen presenting cells following in vivo cDNA immunization.
  • Jurkat- A2.1 K b tumor cells were transfected with pMin.l cDNA and the presentation of the HBY Env 335 epitope by transfected cells was compared to more immunogenic A2.1 -restricted epitopes using specific CTL lines.
  • Epitope presentation was also studied using tumor cells transfected with a control cDNA construct, pMin.2- GFP, that encoded a similar multi-epitope minigene fused with GFP which allows detection of minigene expression in transfected cells by FACS.
  • Epitope presentation of the transfected Jurkat cells was analyzed using specific CTL lines, with cytotoxicity or LFN- ⁇ production serving as a read-out. It was found that the levels of CTL response correlated directly with the in vivo immunogenicity of the epitopes.
  • HBV Env 335-specific CTL line did not recognize the naturally processed epitope efficiently, this line did show an equivalent response to peptide-loaded target cells, as compared to CTL lines specific for the other epitopes ( Figure 23B, D).
  • the immunogenicity of the HBV Env 335 epitope was analyzed to determine whether it may be influenced by its position at the 3' terminus of the minigene construct ( Figure 24D).
  • the position of the Env epitope in the cDNA construct was switched with a more immunogenic epitope, HBV Pol 455, located in the center of the minigene.
  • HBV Pol 455 located in the center of the minigene.
  • this modification also created two potentially new epitopes.
  • the transposition of the two epitopes appeared to affect the immunogenicity of not only the transposed epitopes but also more globally of other epitopes.
  • HLA-A11/K b transgenic mice were immunized to determine whether the three Al 1 epitopes in the pMin.l construct were immunogenic for CTLs. as was the case for the A2.1 -restricted epitopes in the same construct.
  • minigene DNA plasmids can serve as means of delivering multiple epitopes, of varying HLA restrictions and MHC binding affinities, to the immune system in an immunogenic fashion and that appropriate transgenic mouse strains can be used to measure DNA construct immunogenicity in vivo.
  • CTLs were also induced against three Al 1 epitopes in Al 1/K b transgenic mice. These responses suggest that minigene delivery of multiple CTL epitopes that confers broad population coverage may be possible in humans and that transgenic animals of appropriate haplotypes may be a useful tools in optimizing the in vivo immunogenicity of minigene DNA.
  • animals such as monkeys having conserved HLA molecules with cross reactivity to CTL and HTL epitopes recognized by human MHC molecules can be used to determine human immunogenicity of HTL and CTL epitopes (Bertoni et al, J. Immunoll6l :4441-4455 (1998)).
  • HLA transgenic mice represent an attractive alternative, at least for initial vaccine development studies, compared to more cumbersome and expensive studies in higher animal species, such as nonhuman primates.
  • HIV Pol 476 ILKEPVHGV A21 192 2/5 152(29)
  • HBV Core 18 9/9 4555 [22] HIV Env 120 12/12 2119 [37] HBVPol551- ⁇ 9/9 1261 [28] HBV Pol 455 12/12 7386 [13] HIV Pol 476 11/11 7167 [15] HBV Env 335 12/12 437 [18]
  • mice were immunized with pMm 1 DNA or Theradigm-HBV lipopeptide and CTL activity m splenocyte cultures was determined after m vitro stimulation with individual peptide epitopes Results from four independent experiments are shown
  • mice were immunized with pMin.l DNA and CTL activity in splenocyte cultures was determined after in vitro stimulation with individual Al 1 -restricted epitopes. The geometric mean CTL response from three independent experiments are shown.

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Abstract

L'invention concerne des vaccins à base d'acides nucléiques qui codent pour plusieurs épitopes des lymphocytes T cytotoxiques et des lymphocytes T inducteurs ainsi que pour des séquences du C.M.H.
PCT/US1999/010646 1998-05-13 1999-05-13 Vecteurs d'expression destines a stimuler une reponse immunitaire et procedes de leur utilisation WO1999058658A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU40785/99A AU4078599A (en) 1998-05-13 1999-05-13 Expression vectors for stimulating an immune response and methods of using the same
EP99924233A EP1078092B1 (fr) 1998-05-13 1999-05-13 Vecteurs d'expression destines a stimuler une reponse immunitaire et procedes de leur utilisation
JP2000548449A JP2002520000A (ja) 1998-05-13 1999-05-13 免疫応答を刺激するための発現ベクターおよびそのベクターの使用方法
AT99924233T ATE518956T1 (de) 1998-05-13 1999-05-13 Expressionsvektoren zur stimulierung einer immunantwort und verfahren zu deren verwendung
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EP1078092B1 (fr) 2011-08-03
JP2002520000A (ja) 2002-07-09
US20030220285A1 (en) 2003-11-27
US20030216342A1 (en) 2003-11-20
EP1078092A2 (fr) 2001-02-28
CA2331846A1 (fr) 1999-11-18
AU4078599A (en) 1999-11-29
CA2685270C (fr) 2014-07-29
US20030216343A1 (en) 2003-11-20
CA2685270A1 (fr) 1999-11-18
CA2331846C (fr) 2010-01-12
ATE518956T1 (de) 2011-08-15
WO1999058658A3 (fr) 2000-04-20
US6534482B1 (en) 2003-03-18

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